© 2012 Hernández, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**On the Emergence and Evolution**

Additional information is available at the end of the chapter

where translational control plays a prominent role (*4-8*).

Greco Hernández

**1. Introduction**

http://dx.doi.org/10.5772/48568

**of the Eukaryotic Translation Apparatus**

Proteins are one of the elementary components of life and account for a large fraction of mass in the biosphere. They catalyze the big majority of reactions sustaining life, and play structural, transport, and regulatory roles in all living organisms. Hence, "Translation", i.e. the process of decoding a messenger (m)RNA by the ribosome to synthesize a protein, is a fundamental process for all forms of life (*1, 2*). Accordingly, many mechanisms to control gene expression at the translational level have evolved. They allow organisms to *i*) rapidly and reversibly respond to different stresses or sudden environmental changes; *ii*) quickly produce proteins in tissues and developmental processes where transcription is absent or limited; and *iii*) elicit asymmetric localization of proteins when is required (*1-3*). For instance, gametogenesis, early embryogenesis, memory and neurogenesis are processes

The knowledge of the basic processes of translation was established some decades ago, and many regulatory mechanisms have been subsequently elucidated in different organisms (*9- 11*). In recent years, the use of powerful genome-wide sequencing, proteomics and bioinformatics-based technologies in both model and non-model organisms, has shown that a number of components of the translation apparatus has undergone a large diversification across eukaryotes, and that many of them emerged at different times on evolution (*11, 12*). Moreover, universal and lineage-specific mechanisms regulating translation have been described, and evidence supports the notion that some of them might have emerged at different times during evolution (*11, 12*). Evidence supports the notion that some of these mechanisms might have appeared by tinkering, i.e. co-opting and assembling molecules and regulatory mechanisms from other cellular processes (*11*). Overall, the emerging view suggests two general principles. On one hand, that while the fundaments of translation are well conserved across all forms of life, in eukaryotes the initiation step has undergone

## **On the Emergence and Evolution of the Eukaryotic Translation Apparatus**

Greco Hernández

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48568

## **1. Introduction**

Proteins are one of the elementary components of life and account for a large fraction of mass in the biosphere. They catalyze the big majority of reactions sustaining life, and play structural, transport, and regulatory roles in all living organisms. Hence, "Translation", i.e. the process of decoding a messenger (m)RNA by the ribosome to synthesize a protein, is a fundamental process for all forms of life (*1, 2*). Accordingly, many mechanisms to control gene expression at the translational level have evolved. They allow organisms to *i*) rapidly and reversibly respond to different stresses or sudden environmental changes; *ii*) quickly produce proteins in tissues and developmental processes where transcription is absent or limited; and *iii*) elicit asymmetric localization of proteins when is required (*1-3*). For instance, gametogenesis, early embryogenesis, memory and neurogenesis are processes where translational control plays a prominent role (*4-8*).

The knowledge of the basic processes of translation was established some decades ago, and many regulatory mechanisms have been subsequently elucidated in different organisms (*9- 11*). In recent years, the use of powerful genome-wide sequencing, proteomics and bioinformatics-based technologies in both model and non-model organisms, has shown that a number of components of the translation apparatus has undergone a large diversification across eukaryotes, and that many of them emerged at different times on evolution (*11, 12*). Moreover, universal and lineage-specific mechanisms regulating translation have been described, and evidence supports the notion that some of them might have emerged at different times during evolution (*11, 12*). Evidence supports the notion that some of these mechanisms might have appeared by tinkering, i.e. co-opting and assembling molecules and regulatory mechanisms from other cellular processes (*11*). Overall, the emerging view suggests two general principles. On one hand, that while the fundaments of translation are well conserved across all forms of life, in eukaryotes the initiation step has undergone

© 2012 Hernández, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### 32 Cell-Free Protein Synthesis

substantial increase in complexity as compared to prokaryotes (*11, 13-18*); on the other hand, that after eukaryotes emerged the translation apparatus continued evolving to certain degree during eukaryotic diversification (*12*). The continue divergence of eukaryotes led to the diversification of metabolic requirements, to the appearance of different levels of body plans and organismal complexity, and to the arousal of novel developmental programs and behavioural patterns. Altogether, these changes led to the invasion of novel ecological niches. These events most probably were both the causes and effects of a parallel diversification and specialization, to different levels in different taxa, of components and mechanisms of the translation apparatus. Here I will review current knowledge on how this apparatus might have originated and further evolved in eukaryotes, making emphasis on the initiation step of translation.

On the Emergence and Evolution of the Eukaryotic Translation Apparatus 33

Met and GTP) to form a 43S pre-initiation

Met as the

Met in the P-site (*19-22*).

blue) catalyze the binding of amino acids (*aa*) to cognate tRNAs. *Termination* is mediated by the release factors eRF1 (gray) and eRF3 (light blue), and happens when a termination codon (*STOP*) of the mRNA is exposed in the A-site of the ribosome. In this step, the completed polypeptide (blue line) is released. During *Recycling*, which is required to allow further rounds of translation, both ribosomal subunits dissociate from the mRNA. Recycling is assisted by ABCE1 (light blue). eRF1 remains associated with

Translation initiation is mediated by eukaryotic initiation factors (eIF). For the big majority of mRNAs, translation initiation happens by the so-called cap-dependent mechanism (*19- 23*). It begins with the dissociation of the ribosome into its 60S and 40S subunits by eIF6. Free 40S subunit, which is stabilized by eIF3, eIF1 and eIF1A, binds to a ternary complex

complex. In separate events, the cap structure (m7GpppN, where N is any nucleotide) present in the 5' end of the mRNA is recognized by eIF4E in complex with eIF4G (forming the so-called eIF4F complex). Then the 43S pre-initiation complex is recruited to the 5' end of the mRNA, a process that is coordinated by eIF4G through its interactions with eIF4E and the 40S ribosomal subunit-associated eIF3. eIF4G also interacts with the poly A-binding protein (PABP) which interacts with the mRNA 3'-poly(A) tail, thereby promoting circularization of the mRNA and increasing its stability. The closed-loop model proposes that during translation, cross-talk occurs between both mRNA ends due to this circularity. The ribosomal complex then scans in a 5' ⎯> 3' direction along the 5'-untranslated region (UTR) of the mRNA to reach the start codon, usually an AUG. During scanning, eIF4B stimulates the activity of eIF4A, which unwinds secondary RNA structures in the mRNA. eIF1, eIF1A, and eIF5 assist in the positioning of the 40S ribosomal subunit at the correct

start codon so that eIF2 can deliver the anti-codon of the initiator Met-tRNAi

assembled at the start codon of the mRNA containing a Met-tRNAi

initiation of translation (*13, 24, 25*).

**2.2. Elongation**

cognate partner for the start codon, directly to the peptidyl (P)-site of the 40S ribosomal subunit. Once the ribosomal subunit is placed on the start codon, a 48S pre-initiation complex is formed. Then eIF5 promotes GTP hydrolysis by eIF2 to release the eIFs. Finally, the 60S ribosomal subunit joins the 40S subunit in a eIF5B-dependent manner to form an 80S initiation complex. The outcome of the initiation process is a 80S ribosomal complex

In some mRNAs, 5'-UTR recognition by the 40S ribosomal subunit is driven by RNA structures located in *cis* within the mRNA itself. Such structures are defined as Internal Ribosome Entry Site (IRES) and are located nearby the start codon. This mechanism takes place without involvement of the cap structure and eIF4E and is called an IRES-dependent

Translation elongation is assisted by elongation factors (eEF). During this step, mRNA codons are decoded and peptide bonds are formed sequentially to add amino acid residues

the post-termination complexes after polypeptide release.

(consisting of eIF2 bound to an initiator Met-tRNAi

**2.1. Initiation**

## **2. An overview of the translation process in eukaryotes**

Eukaryotic translation is a sophisticated and tightly regulated process, the basic steps of which are conserved in all eukaryotes. It is performed by the ribosome together with multiple 'translation' factors and aminoacyl-tRNA synthetases (aaRSs). It is divided into four major groups of steps: initiation, elongation, termination and recycling (Fig. 1).

**Figure 1.** The general process of translation in eukaryotes. A typical eukaryotic mRNA is represented. The cap structure (m7G), the open reading frame (light blue box) and the poly(A) tail are depicted. During *Initiation*, most eukaryotic mRNAs are translated by the cap-dependent mechanism, which requires recognition by eIF4E (light purple) complexed with eIF4G (red) and eIF4A (light green) –the so-called eIF4F complex– of the cap structure at the 5' end. A 43S pre-initiation complex (consisting in a 40S ribosomal subunit (dark gray) loaded with eIF3 (pink), eIF1 and eIF1A (light grey), initiator MettRNAi Met (blue clover), eIF2 (dark green) and GTP binds the eIF4F-mRNA complex and scans along the 5'-UTR of the mRNA to reach the start codon (usually an *AUG* triplet). During the scanning eIF4A, stimulated by eIF4B (dark blue), unwinds secondary RNA structure in an ATP-dependent manner. The poly A-binding protein (PABP, dark brown) binds both the poly(A) tail and eIF4G promoting mRNA circularization. Free 60S ribosomal subunit is stabilized by eIF6 and eIF3. *Elongation* is assisted by elongation factors eEF1A and eEF2 (light brown). During this step, aminoacyl-tRNA synthetases (aaRSs, blue) catalyze the binding of amino acids (*aa*) to cognate tRNAs. *Termination* is mediated by the release factors eRF1 (gray) and eRF3 (light blue), and happens when a termination codon (*STOP*) of the mRNA is exposed in the A-site of the ribosome. In this step, the completed polypeptide (blue line) is released. During *Recycling*, which is required to allow further rounds of translation, both ribosomal subunits dissociate from the mRNA. Recycling is assisted by ABCE1 (light blue). eRF1 remains associated with the post-termination complexes after polypeptide release.

#### **2.1. Initiation**

32 Cell-Free Protein Synthesis

the initiation step of translation.

tRNAi

substantial increase in complexity as compared to prokaryotes (*11, 13-18*); on the other hand, that after eukaryotes emerged the translation apparatus continued evolving to certain degree during eukaryotic diversification (*12*). The continue divergence of eukaryotes led to the diversification of metabolic requirements, to the appearance of different levels of body plans and organismal complexity, and to the arousal of novel developmental programs and behavioural patterns. Altogether, these changes led to the invasion of novel ecological niches. These events most probably were both the causes and effects of a parallel diversification and specialization, to different levels in different taxa, of components and mechanisms of the translation apparatus. Here I will review current knowledge on how this apparatus might have originated and further evolved in eukaryotes, making emphasis on

Eukaryotic translation is a sophisticated and tightly regulated process, the basic steps of which are conserved in all eukaryotes. It is performed by the ribosome together with multiple 'translation' factors and aminoacyl-tRNA synthetases (aaRSs). It is divided into

**Figure 1.** The general process of translation in eukaryotes. A typical eukaryotic mRNA is represented. The cap structure (m7G), the open reading frame (light blue box) and the poly(A) tail are depicted. During *Initiation*, most eukaryotic mRNAs are translated by the cap-dependent mechanism, which requires recognition by eIF4E (light purple) complexed with eIF4G (red) and eIF4A (light green) –the so-called eIF4F complex– of the cap structure at the 5' end. A 43S pre-initiation complex (consisting in a 40S ribosomal subunit (dark gray) loaded with eIF3 (pink), eIF1 and eIF1A (light grey), initiator Met-

Met (blue clover), eIF2 (dark green) and GTP binds the eIF4F-mRNA complex and scans along the

5'-UTR of the mRNA to reach the start codon (usually an *AUG* triplet). During the scanning eIF4A, stimulated by eIF4B (dark blue), unwinds secondary RNA structure in an ATP-dependent manner. The poly A-binding protein (PABP, dark brown) binds both the poly(A) tail and eIF4G promoting mRNA circularization. Free 60S ribosomal subunit is stabilized by eIF6 and eIF3. *Elongation* is assisted by elongation factors eEF1A and eEF2 (light brown). During this step, aminoacyl-tRNA synthetases (aaRSs,

four major groups of steps: initiation, elongation, termination and recycling (Fig. 1).

**2. An overview of the translation process in eukaryotes**

Translation initiation is mediated by eukaryotic initiation factors (eIF). For the big majority of mRNAs, translation initiation happens by the so-called cap-dependent mechanism (*19- 23*). It begins with the dissociation of the ribosome into its 60S and 40S subunits by eIF6. Free 40S subunit, which is stabilized by eIF3, eIF1 and eIF1A, binds to a ternary complex (consisting of eIF2 bound to an initiator Met-tRNAi Met and GTP) to form a 43S pre-initiation complex. In separate events, the cap structure (m7GpppN, where N is any nucleotide) present in the 5' end of the mRNA is recognized by eIF4E in complex with eIF4G (forming the so-called eIF4F complex). Then the 43S pre-initiation complex is recruited to the 5' end of the mRNA, a process that is coordinated by eIF4G through its interactions with eIF4E and the 40S ribosomal subunit-associated eIF3. eIF4G also interacts with the poly A-binding protein (PABP) which interacts with the mRNA 3'-poly(A) tail, thereby promoting circularization of the mRNA and increasing its stability. The closed-loop model proposes that during translation, cross-talk occurs between both mRNA ends due to this circularity. The ribosomal complex then scans in a 5' ⎯> 3' direction along the 5'-untranslated region (UTR) of the mRNA to reach the start codon, usually an AUG. During scanning, eIF4B stimulates the activity of eIF4A, which unwinds secondary RNA structures in the mRNA. eIF1, eIF1A, and eIF5 assist in the positioning of the 40S ribosomal subunit at the correct start codon so that eIF2 can deliver the anti-codon of the initiator Met-tRNAi Met as the cognate partner for the start codon, directly to the peptidyl (P)-site of the 40S ribosomal subunit. Once the ribosomal subunit is placed on the start codon, a 48S pre-initiation complex is formed. Then eIF5 promotes GTP hydrolysis by eIF2 to release the eIFs. Finally, the 60S ribosomal subunit joins the 40S subunit in a eIF5B-dependent manner to form an 80S initiation complex. The outcome of the initiation process is a 80S ribosomal complex assembled at the start codon of the mRNA containing a Met-tRNAi Met in the P-site (*19-22*).

In some mRNAs, 5'-UTR recognition by the 40S ribosomal subunit is driven by RNA structures located in *cis* within the mRNA itself. Such structures are defined as Internal Ribosome Entry Site (IRES) and are located nearby the start codon. This mechanism takes place without involvement of the cap structure and eIF4E and is called an IRES-dependent initiation of translation (*13, 24, 25*).

#### **2.2. Elongation**

Translation elongation is assisted by elongation factors (eEF). During this step, mRNA codons are decoded and peptide bonds are formed sequentially to add amino acid residues to the carboxy-terminal end of the nascent, mRNA-encoded, peptide (*21, 26-28*). Elongation involves four major steps: *i*) formation of the ternary complex eEF1A⋅GTP⋅aminoacyl-tRNA and delivery of the first elongator aminoacyl-tRNAs to an empty ribosomal tRNA-binding site called the A (acceptor)-site. It is in the A-site where codon/anticodon decoding takes place; *ii*) Interaction of the ribosome with the mRNA-tRNA. This duplex activates eEF1A⋅GTP hydrolysis and guanine nucleotide exchange on eEF1A; *iii*) Peptide bond formation then occurs between the P-site peptidyl-tRNA and the incoming aminoacyl moiety of an A-site aminoacyl-tRNA. This reaction is catalyzed by the peptidyl transferase center of the 60S ribosomal subunit, and the products comprise of a new peptidyl-tRNA that is one amino acid residue longer and a deacylated (discharged) tRNA. *iv*) Binding of eEF2⋅GTP and GTP hydrolysis promote the translocation of the mRNA such that the deacylated tRNA moves to the E (exit)-site, the peptidyl-tRNA is in the P-site, and the mRNA moves by three nucleotides to place the next mRNA codon into the A-site. The deacylated tRNA in E-site is then ejected from the ribosome. The whole process is repeated along the mRNA sequence. When a stop codon is reached the process of termination is initiated (*21, 26-28*).

On the Emergence and Evolution of the Eukaryotic Translation Apparatus 35

The emergence of eukaryotes from prokaryotic ancestors led toward novel, higher levels of cell organization. A plethora of new features emerged at the cellular level, including the acquisition of a nucleus, an endoplasmic reticulocyte and endosymbiotic bacteria, the formation of split genes sorted out in chromosomes, an actin-based cytoskeleton and, in many phyla, the emergence of multicellularity, behavioural patterns and developmental programs. In this new type of cell, novel features appeared in the translation process, including the spatio-temporal separation between transcription and translation, and the increase of the number of events occurring during the initiation step that led to the

During the emergence of eukaryotes, the translation apparatus itself also underwent profound changes, including the evolution of the 40S and 60S ribosomal subunits from the prokaryotic 30S and 50S, respectively. This was due to the addition of several rRNA expansion segments, peptide additions to most ribosomal proteins, and the addition of extra eukaryotic-specific components, including novel ribosomal proteins and the 5.8S rRNA (*26, 35-38*). Moreover, the number of initiation factors increased. While in prokaryotes translation initiation is assisted by three factors, in eukaryotes initiation needs the interplay of at least fourteen factors. Thus, novel, eukaryotic-specific translation factors evolved (including eIF3, eIF4B, eIF4E, eIF4G, eIF4H and eIF5). The mRNA also underwent profound changes which can be summarized as follows: *i*) it acquired a novel molecular structure, from polycistronic to monocistronic, capped, and polyadenylated transcripts; *ii*) it acquired a novel life cycle, from simultaneous transcription/translation, to be transcribed, spliced and exported from the nucleus, to be stored, translated and degraded in the cytoplasm; and *iii*) it acquired a novel functional conformation when engaged in translation, displaying a functional cross-talk between the 5'- and 3'-ends. Finally, with the emergence of eukaryotes the number of different mechanisms that regulate translation was expanded (*11-15, 17, 39*).

How the transition from prokaryotic to eukaryotic translation occurred remains still unresolved. I will discuss some ideas that have been set forward to try to address this

It was established in the 1970s that in eubacteria the recruitment of the small ribosomal subunit to the mRNA occurs by a direct interaction. This happens via the complementary base pairing between the Shine-Dalgarno (SD) sequence of the mRNA, which is a purine-rich region located at around 10 nucleotides upstream the start codon, and a sequence at the 3' end of the 16S rRNA on the ribosome (referred to as anti-Shine-Dalgarno sequence, ASD) (*40, 41*). The importance of the SD sequence to initiate translation was later experimentally corroborated in different eubacteria and archaea (*42-44*), and has been retained in some cell organelles that evolved from eubacteria over a billion years ago (*45*). This, together with the

**4. The transition from prokaryotic to eukaryotic translation initiation**

**4.1. Translation initiation in the prokaryotic world**

**3. The emergence of eukaryotic translation**

establishment of the cap-dependent mechanism.

question.

#### **2.3. Termination**

Translation termination is mediated by two polypeptide chain-release factors, eRF1 and eRF3. When any of the termination codons is exposed in the A-site, eRF1 recognizes the codon, binds the A-site, and triggers the release of the nascent polypeptide from the ribosome by hydrolysing the ester bond linking the polypeptide chain to the P-site tRNA. This reaction leaves the P-site tRNA in a deacylated state, leaving it to be catalyzed by the peptidyl transferase center of the ribosome. eRF1 recognizes stop signals and functionally acts as a tRNA-mimic, whereas eRF3 is a ribosome- and eRF1-dependent GTPase that, by forming a stable complex with eRF1, stimulates the termination process (*21, 29, 30*).

#### **2.4. Recycling**

In the recycling step, both ribosomal subunits are dissociated releasing the mRNA and deacetylated tRNA, so that both ribosomal subunits can be used for another round of initiation (*21, 29, 30*) (Fig.1). Evidence suggests that the ABC-type ATPase ABCE1 is probably the general ribosome recycling factor which coordinates termination with recycling (*31*). ABCE1 establishes multiple contacts with both ribosomal subunits as well as with the release factors and stimulates ribosome dissociation. ABCE1 also influences eRF1 function during stop-codon recognition and peptidyl-tRNA hydrolysis. During ribosome recycling, eRF3 dissociates from ribosomal complexes after GTP hydrolysis, whereas eRF1 remains associated with posttermination ribosomal complexes after peptide release (*31-33*).

According to the closed-loop model, termination and recycling may not release the 40S ribosomal subunit back into the cytoplasm. Instead, this subunit may be passed from the poly(A) tail back to the 5'-end of the mRNA, so that a new round of initiation can be started on the same mRNA (*21, 29, 34*)

## **3. The emergence of eukaryotic translation**

34 Cell-Free Protein Synthesis

initiated (*21, 26-28*).

**2.3. Termination**

**2.4. Recycling**

on the same mRNA (*21, 29, 34*)

to the carboxy-terminal end of the nascent, mRNA-encoded, peptide (*21, 26-28*). Elongation involves four major steps: *i*) formation of the ternary complex eEF1A⋅GTP⋅aminoacyl-tRNA and delivery of the first elongator aminoacyl-tRNAs to an empty ribosomal tRNA-binding site called the A (acceptor)-site. It is in the A-site where codon/anticodon decoding takes place; *ii*) Interaction of the ribosome with the mRNA-tRNA. This duplex activates eEF1A⋅GTP hydrolysis and guanine nucleotide exchange on eEF1A; *iii*) Peptide bond formation then occurs between the P-site peptidyl-tRNA and the incoming aminoacyl moiety of an A-site aminoacyl-tRNA. This reaction is catalyzed by the peptidyl transferase center of the 60S ribosomal subunit, and the products comprise of a new peptidyl-tRNA that is one amino acid residue longer and a deacylated (discharged) tRNA. *iv*) Binding of eEF2⋅GTP and GTP hydrolysis promote the translocation of the mRNA such that the deacylated tRNA moves to the E (exit)-site, the peptidyl-tRNA is in the P-site, and the mRNA moves by three nucleotides to place the next mRNA codon into the A-site. The deacylated tRNA in E-site is then ejected from the ribosome. The whole process is repeated along the mRNA sequence. When a stop codon is reached the process of termination is

Translation termination is mediated by two polypeptide chain-release factors, eRF1 and eRF3. When any of the termination codons is exposed in the A-site, eRF1 recognizes the codon, binds the A-site, and triggers the release of the nascent polypeptide from the ribosome by hydrolysing the ester bond linking the polypeptide chain to the P-site tRNA. This reaction leaves the P-site tRNA in a deacylated state, leaving it to be catalyzed by the peptidyl transferase center of the ribosome. eRF1 recognizes stop signals and functionally acts as a tRNA-mimic, whereas eRF3 is a ribosome- and eRF1-dependent GTPase that, by

In the recycling step, both ribosomal subunits are dissociated releasing the mRNA and deacetylated tRNA, so that both ribosomal subunits can be used for another round of initiation (*21, 29, 30*) (Fig.1). Evidence suggests that the ABC-type ATPase ABCE1 is probably the general ribosome recycling factor which coordinates termination with recycling (*31*). ABCE1 establishes multiple contacts with both ribosomal subunits as well as with the release factors and stimulates ribosome dissociation. ABCE1 also influences eRF1 function during stop-codon recognition and peptidyl-tRNA hydrolysis. During ribosome recycling, eRF3 dissociates from ribosomal complexes after GTP hydrolysis, whereas eRF1 remains associated with posttermination ribosomal complexes after peptide release (*31-33*). According to the closed-loop model, termination and recycling may not release the 40S ribosomal subunit back into the cytoplasm. Instead, this subunit may be passed from the poly(A) tail back to the 5'-end of the mRNA, so that a new round of initiation can be started

forming a stable complex with eRF1, stimulates the termination process (*21, 29, 30*).

The emergence of eukaryotes from prokaryotic ancestors led toward novel, higher levels of cell organization. A plethora of new features emerged at the cellular level, including the acquisition of a nucleus, an endoplasmic reticulocyte and endosymbiotic bacteria, the formation of split genes sorted out in chromosomes, an actin-based cytoskeleton and, in many phyla, the emergence of multicellularity, behavioural patterns and developmental programs. In this new type of cell, novel features appeared in the translation process, including the spatio-temporal separation between transcription and translation, and the increase of the number of events occurring during the initiation step that led to the establishment of the cap-dependent mechanism.

During the emergence of eukaryotes, the translation apparatus itself also underwent profound changes, including the evolution of the 40S and 60S ribosomal subunits from the prokaryotic 30S and 50S, respectively. This was due to the addition of several rRNA expansion segments, peptide additions to most ribosomal proteins, and the addition of extra eukaryotic-specific components, including novel ribosomal proteins and the 5.8S rRNA (*26, 35-38*). Moreover, the number of initiation factors increased. While in prokaryotes translation initiation is assisted by three factors, in eukaryotes initiation needs the interplay of at least fourteen factors. Thus, novel, eukaryotic-specific translation factors evolved (including eIF3, eIF4B, eIF4E, eIF4G, eIF4H and eIF5). The mRNA also underwent profound changes which can be summarized as follows: *i*) it acquired a novel molecular structure, from polycistronic to monocistronic, capped, and polyadenylated transcripts; *ii*) it acquired a novel life cycle, from simultaneous transcription/translation, to be transcribed, spliced and exported from the nucleus, to be stored, translated and degraded in the cytoplasm; and *iii*) it acquired a novel functional conformation when engaged in translation, displaying a functional cross-talk between the 5'- and 3'-ends. Finally, with the emergence of eukaryotes the number of different mechanisms that regulate translation was expanded (*11-15, 17, 39*).

How the transition from prokaryotic to eukaryotic translation occurred remains still unresolved. I will discuss some ideas that have been set forward to try to address this question.

## **4. The transition from prokaryotic to eukaryotic translation initiation**

#### **4.1. Translation initiation in the prokaryotic world**

It was established in the 1970s that in eubacteria the recruitment of the small ribosomal subunit to the mRNA occurs by a direct interaction. This happens via the complementary base pairing between the Shine-Dalgarno (SD) sequence of the mRNA, which is a purine-rich region located at around 10 nucleotides upstream the start codon, and a sequence at the 3' end of the 16S rRNA on the ribosome (referred to as anti-Shine-Dalgarno sequence, ASD) (*40, 41*). The importance of the SD sequence to initiate translation was later experimentally corroborated in different eubacteria and archaea (*42-44*), and has been retained in some cell organelles that evolved from eubacteria over a billion years ago (*45*). This, together with the

#### 36 Cell-Free Protein Synthesis

large proportion of genes having the SD sequence in the well studied bacteria, led to the general idea that for the vast majority of prokaryotic mRNAs the SD sequence was the essential (although not necessarily the sole) element to select the correct initiation codon, and that the SD/ASD interaction during initiation is conserved in most prokaryotes (*16, 46, 47*). However, in recent years a large number of mRNAs lacking a SD sequence have been discovered widespread in a variety of different eubacterial and archaeal lineages. These include mRNAs devoid of 5'-UTR (and hence referred to as "leaderless" mRNAs) (*15, 16, 39, 45, 48-54*), and mRNAs that possess a 5'-UTR and lack a SD sequence (*45, 48-53, 55*). For leaderless mRNAs the start codon itself was found to serve as the most important signal for ribosome recruitment and for translation initiation. Here the initiator tRNA and IF2 are critical for complex formation between the start codon and the ribosome. It is noteworthy that translation initiation of leaderless mRNAs involves the undissociated ribosome 70S instead of the 30S ribosomal subunit (*15, 16, 39, 51, 56-59*). mRNAs with a 5'-UTR devoid of a SD sequence exhibit a pronounced minimum in secondary structure and AUG start codons reside in single-stranded regions of the mRNAs. For these mRNAs, ribosome binding to the start codon is a sequence-independent event, but is strictly dependent on the local absence of RNA secondary (*45*). Translation initiation of these transcripts is promoted by the ribosomal protein S1 (RPS1), which is a component of the 30S ribosomal subunit. RPS1 interacts with the 5'-UTR of an mRNA initiating translation efficiently, regardless of the presence of a SD sequence. Intriguingly, neither archaeobacteria nor eukaryotes contain a RPS1 gene, raising the question of how leadered mRNAs devoid of a SD sequence are translated in Archaea (*39, 46, 49, 51*). Finally, evidence suggests that alternative, unknown mechanisms might be used to initiate translation in Cyanobacteria (*49*) and in haloarchaea (*60*).

On the Emergence and Evolution of the Eukaryotic Translation Apparatus 37

those phyla which lost the SD sequence, RSP1-mediated or leaderless mRNA-used mechanisms of translation initiation work to great extent (*49*). The evolutionary pressures

As mentioned above, while the fundaments of translation are well conserved in all forms of life, in eukaryotes the initiation step underwent substantial increase in complexity and in

Although it is established that eukaryotes evolved from archaeal ancestors, we still don't know what lineage they evolved from. Thus, we don't know what type of mRNA (i.e. SDcontaining, leaderless, or SD-lacking transcript) the first eukaryotes possessed. Nevertheless, all eukaryotic mRNAs lack SD sequences and ribosomes have no RPS1. I have previously suggested that three were the most important evolutionary forces that led to the emergence of the cap-dependent initiation mechanism in eukaryotes, namely (*i*) the need to adjust to the emergence of the nuclear membrane and interrupted genes, (*ii*) the subsequent requirement to splice and export intron-less mRNAs to the cytoplasm, and (*iii*) the absence of SD sequence and RSP1 in eukaryotic mRNAs (*11, 13, 14*). Because eukaryotic mRNAs lack both SD sequences and RPS1 protein, they cannot efficiently recruit the small ribosomal subunit directly to the initiation codon. This, together with the fact that most initiation factors that evolved only within the eukaryotes (including eIF4E, eIF4G, eIF4B, eIF4H and eIF3) are involved in the cap-binding and the scanning processes, indicates that the absence of both SD sequences and RSP1 protein was one of the crucial selection pressures that led to early eukaryotes to develop a novel mechanism to ensure the correct landing of the

ribosome at the 5'-end of mRNAs, namely the cap-dependent initiation (*11, 13, 14*).

I have discussed that during eukaryogenesis and before the time when the cap-dependent initiation was developed, it is possible that there would have been a transition period where the mRNAs of the early eukaryotes were translated in a cap-independent, IRES-driven manner. In this period, 5'-UTRs lacking SD motifs that were able to passively recruit the 40S ribosomal subunit would have been positively selected for and could, therefore, have become the first examples of IRESs (Fig. 2). In this scenario, the cap structure, a proto-eIF4G, the poly(A) tail and an ancestral PABP, might have appeared for functions in RNA metabolism that emerged among the primary adaptive responses to the emergence of split genes and the need for nucleocytoplasmic RNA export, but initially had no role in translation (*11, 13, 14*). As a consequence of the absence of the SD sequence in eukaryotic mRNAs, the scaffold proteins eIF4G, eIF3 and eIF4B, as well as the 5'-end cap structure, eIF4E, and RNA helicases were later incorporated into the already established but incipient eukaryotic translation machinery because they ensured a more efficient recruitment of the 40S ribosomal subunit by the mRNA. Altogether, these events led the "scanning" process to evolve and to the establishment of the current cap-dependent translation initiation. Mutations in PABP, which allowed binding to eIF4G and promoted mRNA circularization underwent a strong positive selection because they ensured a more efficient recruitment of

that led to the loss of SD sequences, however, are completely unknown.

number of initiation factors as compared to prokaryotes.

**4.3. The transition to eukaryotes**

Overall, the emerging view indicates that in the prokaryotic world, both SD-dependent and SD-independent translation mechanisms are present in all major groups of prokaryotes. Indeed, evidence suggests that the leaderless mechanism might represent the major pathway to initiate translation in Archaea (*52-54*). Thus, it has been suggested that the last common ancestor of existing life already possessed an established fundamental translational apparatus, but the mechanisms to initiate translation initiation further diversified in the bacterial and archaeal lineages (*17, 49, 50, 60*).

#### **4.2. What was the mechanism to recruit mRNAs in the last common ancestor of existing life?**

Despite the presence of leaderless and leadered, SD-lacking mRNAs across prokaryotes, a recent study using the genomes of 277 prokaryote species, both eubacteria and archaea, showed that the anti-SD sequence at the 3' end of the 16S rRNA on the ribosome is highly conserved among all species, and that loss of the SD sequence seems to have occurred multiple times, independently, in different phyla (*49*). These observations strongly suggest that the SD/ASD interaction plays an important role in translation initiation in essentially all prokaryote species that are descended from the last universal common ancestor. Thus, the SD-based mechanism of ribosome recruiting might have driven translation initiation in the last common ancestor of existing organisms, but was further lost in different phyla (*49*). For those phyla which lost the SD sequence, RSP1-mediated or leaderless mRNA-used mechanisms of translation initiation work to great extent (*49*). The evolutionary pressures that led to the loss of SD sequences, however, are completely unknown.

#### **4.3. The transition to eukaryotes**

36 Cell-Free Protein Synthesis

large proportion of genes having the SD sequence in the well studied bacteria, led to the general idea that for the vast majority of prokaryotic mRNAs the SD sequence was the essential (although not necessarily the sole) element to select the correct initiation codon, and that the SD/ASD interaction during initiation is conserved in most prokaryotes (*16, 46, 47*). However, in recent years a large number of mRNAs lacking a SD sequence have been discovered widespread in a variety of different eubacterial and archaeal lineages. These include mRNAs devoid of 5'-UTR (and hence referred to as "leaderless" mRNAs) (*15, 16, 39, 45, 48-54*), and mRNAs that possess a 5'-UTR and lack a SD sequence (*45, 48-53, 55*). For leaderless mRNAs the start codon itself was found to serve as the most important signal for ribosome recruitment and for translation initiation. Here the initiator tRNA and IF2 are critical for complex formation between the start codon and the ribosome. It is noteworthy that translation initiation of leaderless mRNAs involves the undissociated ribosome 70S instead of the 30S ribosomal subunit (*15, 16, 39, 51, 56-59*). mRNAs with a 5'-UTR devoid of a SD sequence exhibit a pronounced minimum in secondary structure and AUG start codons reside in single-stranded regions of the mRNAs. For these mRNAs, ribosome binding to the start codon is a sequence-independent event, but is strictly dependent on the local absence of RNA secondary (*45*). Translation initiation of these transcripts is promoted by the ribosomal protein S1 (RPS1), which is a component of the 30S ribosomal subunit. RPS1 interacts with the 5'-UTR of an mRNA initiating translation efficiently, regardless of the presence of a SD sequence. Intriguingly, neither archaeobacteria nor eukaryotes contain a RPS1 gene, raising the question of how leadered mRNAs devoid of a SD sequence are translated in Archaea (*39, 46, 49, 51*). Finally, evidence suggests that alternative, unknown mechanisms might be used

to initiate translation in Cyanobacteria (*49*) and in haloarchaea (*60*).

bacterial and archaeal lineages (*17, 49, 50, 60*).

**existing life?**

Overall, the emerging view indicates that in the prokaryotic world, both SD-dependent and SD-independent translation mechanisms are present in all major groups of prokaryotes. Indeed, evidence suggests that the leaderless mechanism might represent the major pathway to initiate translation in Archaea (*52-54*). Thus, it has been suggested that the last common ancestor of existing life already possessed an established fundamental translational apparatus, but the mechanisms to initiate translation initiation further diversified in the

**4.2. What was the mechanism to recruit mRNAs in the last common ancestor of**

Despite the presence of leaderless and leadered, SD-lacking mRNAs across prokaryotes, a recent study using the genomes of 277 prokaryote species, both eubacteria and archaea, showed that the anti-SD sequence at the 3' end of the 16S rRNA on the ribosome is highly conserved among all species, and that loss of the SD sequence seems to have occurred multiple times, independently, in different phyla (*49*). These observations strongly suggest that the SD/ASD interaction plays an important role in translation initiation in essentially all prokaryote species that are descended from the last universal common ancestor. Thus, the SD-based mechanism of ribosome recruiting might have driven translation initiation in the last common ancestor of existing organisms, but was further lost in different phyla (*49*). For As mentioned above, while the fundaments of translation are well conserved in all forms of life, in eukaryotes the initiation step underwent substantial increase in complexity and in number of initiation factors as compared to prokaryotes.

Although it is established that eukaryotes evolved from archaeal ancestors, we still don't know what lineage they evolved from. Thus, we don't know what type of mRNA (i.e. SDcontaining, leaderless, or SD-lacking transcript) the first eukaryotes possessed. Nevertheless, all eukaryotic mRNAs lack SD sequences and ribosomes have no RPS1. I have previously suggested that three were the most important evolutionary forces that led to the emergence of the cap-dependent initiation mechanism in eukaryotes, namely (*i*) the need to adjust to the emergence of the nuclear membrane and interrupted genes, (*ii*) the subsequent requirement to splice and export intron-less mRNAs to the cytoplasm, and (*iii*) the absence of SD sequence and RSP1 in eukaryotic mRNAs (*11, 13, 14*). Because eukaryotic mRNAs lack both SD sequences and RPS1 protein, they cannot efficiently recruit the small ribosomal subunit directly to the initiation codon. This, together with the fact that most initiation factors that evolved only within the eukaryotes (including eIF4E, eIF4G, eIF4B, eIF4H and eIF3) are involved in the cap-binding and the scanning processes, indicates that the absence of both SD sequences and RSP1 protein was one of the crucial selection pressures that led to early eukaryotes to develop a novel mechanism to ensure the correct landing of the ribosome at the 5'-end of mRNAs, namely the cap-dependent initiation (*11, 13, 14*).

I have discussed that during eukaryogenesis and before the time when the cap-dependent initiation was developed, it is possible that there would have been a transition period where the mRNAs of the early eukaryotes were translated in a cap-independent, IRES-driven manner. In this period, 5'-UTRs lacking SD motifs that were able to passively recruit the 40S ribosomal subunit would have been positively selected for and could, therefore, have become the first examples of IRESs (Fig. 2). In this scenario, the cap structure, a proto-eIF4G, the poly(A) tail and an ancestral PABP, might have appeared for functions in RNA metabolism that emerged among the primary adaptive responses to the emergence of split genes and the need for nucleocytoplasmic RNA export, but initially had no role in translation (*11, 13, 14*). As a consequence of the absence of the SD sequence in eukaryotic mRNAs, the scaffold proteins eIF4G, eIF3 and eIF4B, as well as the 5'-end cap structure, eIF4E, and RNA helicases were later incorporated into the already established but incipient eukaryotic translation machinery because they ensured a more efficient recruitment of the 40S ribosomal subunit by the mRNA. Altogether, these events led the "scanning" process to evolve and to the establishment of the current cap-dependent translation initiation. Mutations in PABP, which allowed binding to eIF4G and promoted mRNA circularization underwent a strong positive selection because they ensured a more efficient recruitment of the 40S ribosomal subunit by the mRNA, stimulating translation and increasing mRNA stability. mRNA circularization provides an effective means for the protein synthesis apparatus to selectively translate only intact mRNAs, i.e., those that harbor both a cap and a poly(A) tail. Thus, mRNA circularization also (and perhaps primarily) underwent a strong positive selection because it represents a checkpoint that determines whether or not to initiate translation (*11, 13, 14*).

On the Emergence and Evolution of the Eukaryotic Translation Apparatus 39

**5. Diversification of eukaryotes and further evolution of the translation**

After eukaryotes emerged, components of the translation machinery further evolved along the radiation into different phyla (Fig. 3). The continue appearance of different levels of organismal complexity led to the arousal of new phyla, developmental programs, behavioral patterns, and the invasion of novel ecological niches by eukaryotes. These events most probably were the causes of a further evolution, to different levels, of components and mechanisms of the translation apparatus in different taxa (*11, 12*). In the following, I will

**Figure 3.** Diversity in the components of the translation apparatus across eukaryotes. The different components of the translation machinery with well-studied diversity in different phyla are shown in colors. Components with some diversity that is not discussed here are depicted in gray. Several cognates of eIF4E (purple) and eIF4G (red) have been found in plants, metazoan, protists and some fungi. In some cases, eIF4E cognates have evolved towards translational repressors (4E-HP, dark blue, is an example). Many 4E-binding proteins (including Maskin, 4E-BPs, Eap1, p20 and Cup, light blue) have been discovered in different species. The subunit composition of eIF3 (pink) ranges from 5 to 13 nonidentical polypeptides in different phyla. There is, however, a core of five homolog subunits shared by most eukaryotes. Some diversity has been found in eIF6. Several RNA helicases (light green) from diverse organisms are involved in the *Initiation* step. A family of five kinases (*HRI*, *PERK*, *GCN2*, *PKR* and *PKZ*, red) phosphorylate the alpha subunit of eIF2 to inhibit global translation under stress conditions. The presence of eIF2alpha kinases varies in different lineages. Different domains, such as *WHEP*, *EMAPII*, and *UNE-S*, have been added to different aminoacyl-tRNA synthetases (*aaRSs*, blue) in distinct phyla of multicellular species. For *Elongation* to happen, a number of protist, algae and fungi lack eEF1A (light brown) and instead possess the related factor elongation factor-like (EFL, dark brown). Ribosomes from all eukaryotes perform *Elongation* with eEF1A and eEF2. However, the yeast *S. cerevisiae* requires an additional essential factor, eEF3 (dark pink), for *Elongation* to proceed. Genes encoding eEF3 have been found exclusively in many species of fungi. Evidence supports the notion that eEF3 activity promotes ribosome recycling. Several cytoplasmic PABP (dark brown) cognates have been found in many phyla.

**5.1. Functional divergence of initiation factors**

summarize the most studied examples of this.

**apparatus**

initiation of translation. (A) Ancestral archaeal cells had polycistronic (blue boxes) mRNAs. It is not known what was the mechanism used by the prokaryotic ancestors of eukaryotes to recruit the mRNA by the 30S ribosomal subunit. (B) The evolutionary transition from ancestral archaeal cells to early eukaryotes is represented. Due to the appearance of the nucleus and split genes, transcription and translation were decoupled, and a need to splice, export and protect the transcripts during nucleuscytoplasm export emerged. The cap structure might have first appeared at this evolutionary stage to provide a "platform" to assemble the splicing, export and RNA protection mechanisms (see reference *14* for details). The arousal of PABP might have happened at this stage as part of the mechanisms to protect mRNAs (see reference *11* for details). The lack of SD sequences in the mRNAs, probably as a result of massive invasion of introns from endosimbionts, as well as the apparition of monocistronic mRNAs and both the 40S and the 60S ribosomal subunits, happened at this stage. Initiation of translation occurred perhaps in an IRES-dependent manner via the direct recruitment of the 40S ribosomal subunit by the mRNA. (C) The evolution in the cytoplasm of a proto-eIF4G (Pr-4G), along with the emergence of eIF3 and eIF4B, gradually improved the delivery of the 40S ribosomal subunit to the early mRNAs during evolution. The scanning process (orange arrow) evolved probably at this stage due to the activity of RNA helicases coming from different processes of metabolism that at the same time could participate in the unwinding of mRNA secondary structure during translation initiation. Among them, however, only an eIF4A-like helicase evolved functional interactions with eIF4G thus becoming later the canonical initiation factor eIF4A. (D) It is not know when eIF4E evolved during eukaryogenesis. However, an interaction of eIF4G with eIF4E, eIF4A and PABP evolved, which eventually led to the establishment of today's widespread cap-dependent mechanism to initiate translation.

## **5. Diversification of eukaryotes and further evolution of the translation apparatus**

#### **5.1. Functional divergence of initiation factors**

38 Cell-Free Protein Synthesis

initiate translation (*11, 13, 14*).

**Figure 2.** A proposed model for the evolution of the cap-dependent.

today's widespread cap-dependent mechanism to initiate translation.

initiation of translation. (A) Ancestral archaeal cells had polycistronic (blue boxes) mRNAs. It is not known what was the mechanism used by the prokaryotic ancestors of eukaryotes to recruit the mRNA by

the 30S ribosomal subunit. (B) The evolutionary transition from ancestral archaeal cells to early eukaryotes is represented. Due to the appearance of the nucleus and split genes, transcription and translation were decoupled, and a need to splice, export and protect the transcripts during nucleuscytoplasm export emerged. The cap structure might have first appeared at this evolutionary stage to provide a "platform" to assemble the splicing, export and RNA protection mechanisms (see reference *14* for details). The arousal of PABP might have happened at this stage as part of the mechanisms to protect mRNAs (see reference *11* for details). The lack of SD sequences in the mRNAs, probably as a result of massive invasion of introns from endosimbionts, as well as the apparition of monocistronic mRNAs and both the 40S and the 60S ribosomal subunits, happened at this stage. Initiation of translation occurred perhaps in an IRES-dependent manner via the direct recruitment of the 40S ribosomal subunit by the mRNA. (C) The evolution in the cytoplasm of a proto-eIF4G (Pr-4G), along with the emergence of eIF3 and eIF4B, gradually improved the delivery of the 40S ribosomal subunit to the early mRNAs during evolution. The scanning process (orange arrow) evolved probably at this stage due to the activity of RNA helicases coming from different processes of metabolism that at the same time could participate in the unwinding of mRNA secondary structure during translation initiation. Among them, however, only an eIF4A-like helicase evolved functional interactions with eIF4G thus becoming later the canonical initiation factor eIF4A. (D) It is not know when eIF4E evolved during eukaryogenesis. However, an interaction of eIF4G with eIF4E, eIF4A and PABP evolved, which eventually led to the establishment of

the 40S ribosomal subunit by the mRNA, stimulating translation and increasing mRNA stability. mRNA circularization provides an effective means for the protein synthesis apparatus to selectively translate only intact mRNAs, i.e., those that harbor both a cap and a poly(A) tail. Thus, mRNA circularization also (and perhaps primarily) underwent a strong positive selection because it represents a checkpoint that determines whether or not to

After eukaryotes emerged, components of the translation machinery further evolved along the radiation into different phyla (Fig. 3). The continue appearance of different levels of organismal complexity led to the arousal of new phyla, developmental programs, behavioral patterns, and the invasion of novel ecological niches by eukaryotes. These events most probably were the causes of a further evolution, to different levels, of components and mechanisms of the translation apparatus in different taxa (*11, 12*). In the following, I will summarize the most studied examples of this.

**Figure 3.** Diversity in the components of the translation apparatus across eukaryotes. The different components of the translation machinery with well-studied diversity in different phyla are shown in colors. Components with some diversity that is not discussed here are depicted in gray. Several cognates of eIF4E (purple) and eIF4G (red) have been found in plants, metazoan, protists and some fungi. In some cases, eIF4E cognates have evolved towards translational repressors (4E-HP, dark blue, is an example). Many 4E-binding proteins (including Maskin, 4E-BPs, Eap1, p20 and Cup, light blue) have been discovered in different species. The subunit composition of eIF3 (pink) ranges from 5 to 13 nonidentical polypeptides in different phyla. There is, however, a core of five homolog subunits shared by most eukaryotes. Some diversity has been found in eIF6. Several RNA helicases (light green) from diverse organisms are involved in the *Initiation* step. A family of five kinases (*HRI*, *PERK*, *GCN2*, *PKR* and *PKZ*, red) phosphorylate the alpha subunit of eIF2 to inhibit global translation under stress conditions. The presence of eIF2alpha kinases varies in different lineages. Different domains, such as *WHEP*, *EMAPII*, and *UNE-S*, have been added to different aminoacyl-tRNA synthetases (*aaRSs*, blue) in distinct phyla of multicellular species. For *Elongation* to happen, a number of protist, algae and fungi lack eEF1A (light brown) and instead possess the related factor elongation factor-like (EFL, dark brown). Ribosomes from all eukaryotes perform *Elongation* with eEF1A and eEF2. However, the yeast *S. cerevisiae* requires an additional essential factor, eEF3 (dark pink), for *Elongation* to proceed. Genes encoding eEF3 have been found exclusively in many species of fungi. Evidence supports the notion that eEF3 activity promotes ribosome recycling. Several cytoplasmic PABP (dark brown) cognates have been found in many phyla.

Most evidence for molecular and functional diversification among the translation components has been found in the eIF4 proteins. All major eukaryotic lineages possess several paralog genes for members of the eIF4 families. For some of them, differential expression patterns, and even variable biochemical properties among paralogs of the same organism, have been found (*12, 14, 61-66*). For eIF4E and eIF4G cognates, physiological specialization has been also found and, in some cases, eIF4E cognates have evolved towards translational repressors (*12, 14, 61-63, 65*). These findings support the hypothesis that in organisms with several paralogs, a ubiquitous set of eIF4 factors supports global translation initiation whereas other paralogs perform their activity in specific cellular processes (*61*). Whereas the need for distinct eIF4 proteins in different tissues may have been the driving force behind the evolution of various paralogs in multicellular organisms, in unicellular eukaryotes different paralogs may be differentially needed during distinct life stages (*67*).The multisubunit eIF3 is another example of factor that has undergone molecular diversification across eukaryotes, whose subunit composition ranges from 5 to 13 nonidentical polypeptides in different phyla (*68*). However, the functional relevance of these phenomena is not known.

On the Emergence and Evolution of the Eukaryotic Translation Apparatus 41

single duplication event in a specific lineage gave rise to *EFL*. *EFL* genes were then spread to other lineages via multiple independent lateral gene transfer events, where EFL took over the original eEF1A function resulting in secondary loss of the endogenous *eEF1A*. It is thought that both genes co-existed for some time before one or the other was lost. Indeed, the diatom *Thalassiosira* bears both *EFL* and *eEF1A* genes (*79*) and might be an example of this situation. It is also possible that there was a single gain of *EFL* early in evolution followed by differential loss of it (*74, 78, 79, 81, 82*). So far, *EFL* genes have been identified in widespread taxa, including diatoms, green and red algae, fungi, euglenozoans, foraminiferans, cryptophytes, goniomonads, katablepharid, chlorarachniophytes, oomycetes, dinoflagellates, choanozoans, centrohelids and haptophytes. Most of them are unicellular organisms. In contrast, most

Key molecules for elongation are aaRSs, which catalyze the aminoacylation reaction whereby an amino acid is attached to the cognate tRNA (*21, 26, 27*). aaRS represent an intriguing and perhaps unique case of evolution among the components of the translation apparatus. Throughout evolution of multicellularity, different domains such as the tripeptide ELR (Glu-Leu-Arg), the oligonucleotide binding fold-containing EMAPII domain, the WHEP domain, the glutathione S-transferase (GST) domain and a specialized aminoterminal helix (N-helix), have been progressively added to different aaRSs in distinct phyla. The tripeptide ELR and the EMAPII domain were incorporated simultaneously to TyrRSs in metazoans starting from insects; the WHEP domain is present in TrpRS only in chordates; and a unique sequence motif, UNE-S, became fused to the C-terminal of SerRS of all vertebrates. In bilaterian the glutamylRS and prolylRS were linked via WHEP domains

It has been found that the function of the aaRSs was either increased or impaired by the addition of the new domains. Whereas the WHEP domain regulates interaction of TrpRS with its cognate receptor, with MetRS this domain plays a tRNA-sequestering function. The Leu-zipper motif in ArgRS is important for the formation of multi aaRSs complex (MSC), which enhances channeling of tRNA to the ribosome. Moreover, different aaRSs play diverse roles in cellular activities beyond translation, such as stress response, plant and animal embryogenesis, cell death, immune responses, transcriptional regulation, and RNA splicing (*83-85*). It has been found that the incorporation of domains to aaRSs correlates positively with the increase in organism's complexity. For example, the number of aaRSs carrying the GST domain increases from two in fungi to four in insects, to five in fish and six in humans (*83*). Thus, it has been proposed that the newly fused aaRSs domains triggered the appearance of new biological functions for these proteins in different lineages, and that the fusion of domains to aaRSs could have played an important part in expanding the

Termination is governed by eRF1, which is a monophyletic protein that was inherited by eukaryotes from archaeal ancestors. eRF1 is universally present in eukaryotes and, with the

eukaryotes contain only *eEF1A* (*74-82*).

giving rise to a bifunctional glutamyl-prolylRS (*83, 84*).

complexity of newly emerging metazoan phyla (*83*).

**5.4. Divergence in termination and recycling factors**

#### **5.2. Multiple helicases involved in the initiation step**

eIF4A is the factor traditionally thought to perform RNA helicase activity to unwind the 5'- UTR secondary structure during the scanning. Recently, other RNA helicases from diverse organisms have also been found to be involved in different steps of translation initiation. Such is the case of the mammalian, *Drosophila* and yeast helicases DDX3 and Ded1, as well as the human helicases RHA and DHX29 (*69-71*). Evidence supports the idea that in *Drosophila*, the helicase Vasa is a translational activator of specific mRNAs involved in germline development (*5, 6*). In contrast, orthologs of the *Xenopus* helicase Xp54 in several organisms, including *Drosophila* Me31B, *Saccharomyces* Dhh1, human rck/p54, and *Caenorhabditis* CGH-1 have been found to repress translation of stored mRNAs and promote aggregation into germplasm-containing structures (*72*). In *Arabidopsis*, the eIF4F complex contains eIF4A in proliferating cells but different RNA helicases in quiescent cells (*73*). These findings show that evolutionary convergence has happened in different lineages to fulfill the need of RNA helicase activity during translation initiation.

#### **5.3. Divergence in molecules involved in the elongation step**

In contrast to the initiation step, the process of elongation is highly conserved among all forms of life. Strikingly, a recent genome-wide analysis revealed that a number of eukaryotic lineages lack eEF1A, a canonical factor that delivers aa-tRNAs to the A-site of ribosomes during the elongation step. Instead, they possess a related factor called elongation factor-like (EFL) protein that retains the residues critical for eEF1A (*74*). It was later found that EFL-encoding species are scattered widely across eukaryotes and that *eEF1A* and *EFL* genes display mutually exclusive phylogenetic distributions. Thus, it is assumed that eEF1A and EFL are functionally equivalent (*74-82*). It is thought that *eEF1A* is ancestral to all extant eukaryotes and that a single duplication event in a specific lineage gave rise to *EFL*. *EFL* genes were then spread to other lineages via multiple independent lateral gene transfer events, where EFL took over the original eEF1A function resulting in secondary loss of the endogenous *eEF1A*. It is thought that both genes co-existed for some time before one or the other was lost. Indeed, the diatom *Thalassiosira* bears both *EFL* and *eEF1A* genes (*79*) and might be an example of this situation. It is also possible that there was a single gain of *EFL* early in evolution followed by differential loss of it (*74, 78, 79, 81, 82*). So far, *EFL* genes have been identified in widespread taxa, including diatoms, green and red algae, fungi, euglenozoans, foraminiferans, cryptophytes, goniomonads, katablepharid, chlorarachniophytes, oomycetes, dinoflagellates, choanozoans, centrohelids and haptophytes. Most of them are unicellular organisms. In contrast, most eukaryotes contain only *eEF1A* (*74-82*).

40 Cell-Free Protein Synthesis

phenomena is not known.

**5.2. Multiple helicases involved in the initiation step**

fulfill the need of RNA helicase activity during translation initiation.

**5.3. Divergence in molecules involved in the elongation step**

Most evidence for molecular and functional diversification among the translation components has been found in the eIF4 proteins. All major eukaryotic lineages possess several paralog genes for members of the eIF4 families. For some of them, differential expression patterns, and even variable biochemical properties among paralogs of the same organism, have been found (*12, 14, 61-66*). For eIF4E and eIF4G cognates, physiological specialization has been also found and, in some cases, eIF4E cognates have evolved towards translational repressors (*12, 14, 61-63, 65*). These findings support the hypothesis that in organisms with several paralogs, a ubiquitous set of eIF4 factors supports global translation initiation whereas other paralogs perform their activity in specific cellular processes (*61*). Whereas the need for distinct eIF4 proteins in different tissues may have been the driving force behind the evolution of various paralogs in multicellular organisms, in unicellular eukaryotes different paralogs may be differentially needed during distinct life stages (*67*).The multisubunit eIF3 is another example of factor that has undergone molecular diversification across eukaryotes, whose subunit composition ranges from 5 to 13 nonidentical polypeptides in different phyla (*68*). However, the functional relevance of these

eIF4A is the factor traditionally thought to perform RNA helicase activity to unwind the 5'- UTR secondary structure during the scanning. Recently, other RNA helicases from diverse organisms have also been found to be involved in different steps of translation initiation. Such is the case of the mammalian, *Drosophila* and yeast helicases DDX3 and Ded1, as well as the human helicases RHA and DHX29 (*69-71*). Evidence supports the idea that in *Drosophila*, the helicase Vasa is a translational activator of specific mRNAs involved in germline development (*5, 6*). In contrast, orthologs of the *Xenopus* helicase Xp54 in several organisms, including *Drosophila* Me31B, *Saccharomyces* Dhh1, human rck/p54, and *Caenorhabditis* CGH-1 have been found to repress translation of stored mRNAs and promote aggregation into germplasm-containing structures (*72*). In *Arabidopsis*, the eIF4F complex contains eIF4A in proliferating cells but different RNA helicases in quiescent cells (*73*). These findings show that evolutionary convergence has happened in different lineages to

In contrast to the initiation step, the process of elongation is highly conserved among all forms of life. Strikingly, a recent genome-wide analysis revealed that a number of eukaryotic lineages lack eEF1A, a canonical factor that delivers aa-tRNAs to the A-site of ribosomes during the elongation step. Instead, they possess a related factor called elongation factor-like (EFL) protein that retains the residues critical for eEF1A (*74*). It was later found that EFL-encoding species are scattered widely across eukaryotes and that *eEF1A* and *EFL* genes display mutually exclusive phylogenetic distributions. Thus, it is assumed that eEF1A and EFL are functionally equivalent (*74-82*). It is thought that *eEF1A* is ancestral to all extant eukaryotes and that a Key molecules for elongation are aaRSs, which catalyze the aminoacylation reaction whereby an amino acid is attached to the cognate tRNA (*21, 26, 27*). aaRS represent an intriguing and perhaps unique case of evolution among the components of the translation apparatus. Throughout evolution of multicellularity, different domains such as the tripeptide ELR (Glu-Leu-Arg), the oligonucleotide binding fold-containing EMAPII domain, the WHEP domain, the glutathione S-transferase (GST) domain and a specialized aminoterminal helix (N-helix), have been progressively added to different aaRSs in distinct phyla. The tripeptide ELR and the EMAPII domain were incorporated simultaneously to TyrRSs in metazoans starting from insects; the WHEP domain is present in TrpRS only in chordates; and a unique sequence motif, UNE-S, became fused to the C-terminal of SerRS of all vertebrates. In bilaterian the glutamylRS and prolylRS were linked via WHEP domains giving rise to a bifunctional glutamyl-prolylRS (*83, 84*).

It has been found that the function of the aaRSs was either increased or impaired by the addition of the new domains. Whereas the WHEP domain regulates interaction of TrpRS with its cognate receptor, with MetRS this domain plays a tRNA-sequestering function. The Leu-zipper motif in ArgRS is important for the formation of multi aaRSs complex (MSC), which enhances channeling of tRNA to the ribosome. Moreover, different aaRSs play diverse roles in cellular activities beyond translation, such as stress response, plant and animal embryogenesis, cell death, immune responses, transcriptional regulation, and RNA splicing (*83-85*). It has been found that the incorporation of domains to aaRSs correlates positively with the increase in organism's complexity. For example, the number of aaRSs carrying the GST domain increases from two in fungi to four in insects, to five in fish and six in humans (*83*). Thus, it has been proposed that the newly fused aaRSs domains triggered the appearance of new biological functions for these proteins in different lineages, and that the fusion of domains to aaRSs could have played an important part in expanding the complexity of newly emerging metazoan phyla (*83*).

#### **5.4. Divergence in termination and recycling factors**

Termination is governed by eRF1, which is a monophyletic protein that was inherited by eukaryotes from archaeal ancestors. eRF1 is universally present in eukaryotes and, with the

#### 42 Cell-Free Protein Synthesis

exception of some vascular plants and some ciliates, eukaryotes contain only one *eRF1* gene (*86-89*). Interestingly, unusually high rates of eRF1 evolution have been found in organisms with variant genetic codes (mostly protists and unicellular fungi), especially in the Nterminal domain, which is responsible for stop-codon recognition (*86, 87, 89-92*). eRF1 displays structural similarity to tRNA molecules and mimics its activity during binding of ribosomal A-site during recognition of a stop codon (*91-94*). Since mutations in eRF1 Nterminal domain switch from omnipotent to bipotent mode for stop-codon specificity (*94- 98*), most likely the accelerated evolution of eRF1 in organisms with variations to the nuclear genetic code has been driven mainly to accommodate these variations (*89-99*).

On the Emergence and Evolution of the Eukaryotic Translation Apparatus 43

apparatus also diverged (*108*). Another fundamental aspect of RNA metabolism is the storage and degradation of mRNAs in different cytoplasmic bodies, such as P-bodies and Pgranules, which contain translation factors (*109*). The diversity and conservation of these foci across phyla are a reflection of the general evolution that the translation machinery and

*Division of Basic Research, National Institute for Cancer (INCan), Tlalpan, Mexico City, Mexico*

I was supported by the National Institute for Cancer (Instituto Nacional de Cancerología,

[1] Mathews, M. B., Sonenberg, N., and Hershey, J. W. B. (2000) Origins and principles of translational control, in *Translational control of gene expression* (Sonenberg, N., Hershey, J. W. B., and Mathews, M. B., Eds.), pp 1-31, Cold Spring Harbor Laboratory press, Cold

[2] Mathews, M. B., Sonenberg, N., and Hershey, J. W. B. (2007) Origins and principles of translational control, in *Translational control in biology and medicine* (Mathews, M. B., Sonenberg, N., and Hershey, J. W. B., Eds.), pp 1-40, Cold Spring Harbor Laboratory

[3] Mazumder, B., Seshadri, V., and Fox, P. L. (2003) Translational control by the 3'-UTR:

[4] Renkawitz-Pohl, R., Hempel, L., Hollman, M., and Schafer, M. A. (2005) Spermatogenesis, in *Comprehensive molecular insect science* (Gilbert, L. I., Iatrou, K., and

[5] Richter, J. D., and Lasko, P. (2011) Translational control in oocyte development, *Cold*

[6] Lasko, P. (2009) Translational control during early development, in *Progress in Molecular Biology and Translational Science* (Hershey, J. W. B., Ed.), pp 211-254, Academic Press,

[7] Thompson, B., Wickens, M., and Kimble, J. (2007) Translational control in development, in *Translational control in biology and medicine* (Mathews, M. B., Sonenberg, N., and Hershey, J. W. B., Eds.), pp 507-544, Cold Spring Harbor Laboratory Press, Cold Spring

[8] Sonenberg, N., and Hinnebusch, A. G. (2007) New modes of translation control in

its regulation have undergone during eukaryotes evolution.

**Author details**

Greco Hernández

México).

**7. References**

Burlington.

Harbor, New York.

**Acknowledgement**

Spring Harbor, New York.

Press, Cold Spring Harbor, New York.

*Spring Harbor Perspect. Biol. 3*, a002758.

the ends specify the means. *Trends Biochem. Sci. 28*, 91-98.

development, behavior, and disease, *Mol. Cell 28*, 721-729.

Gill, S. S., Eds.), pp 157-177, Elsevier Pergamon.

Another striking case of evolutionary divergence was found in the fungi. Ribosomes from all eukaryotes perform elongation with eEF1A and eEF2. Surprisingly, it was found that the yeast *Saccharomyces cerevisiae* requires an additional essential factor, eEF3, for the elongation cycle to proceed (*100*). Genes encoding eEF3 were subsequently identified exclusively in other fungi, including *Candida*, *Pneumocystis*, *Neurospora*, *Aspergillus* and *Mucor* (*101-104*). eEF3 is an ATPase that interacts with both ribosomal subunits and that is required for the binding of aminoacyl-tRNA-eEF1A-GTP ternary complex to the ribosomal A-site by enhancing the rate of deacylated tRNA dissociation from the E-site (*105*). Recently, it was shown that post-termination complex, consisting of a ribosome, mRNA, and tRNA, is disassembled into single components by ATP and eEF3. Because the release of mRNA and deacylated tRNA and ribosome dissociation takes place simultaneously and no 40S⎯mRNA complexes remain, it is proposed that eEF3 activity promotes ribosome recycling (*106*). It remains unsolved what were the evolutionary forces that led to the emergence of eEF3 exclusively in fungi.

## **6. Concluding remarks**

One of the enigmas in current Biology is how eukaryotic protein synthesis emerged. I have discussed that, in the absence of SD sequence in mRNAs and RPS1 in ribosomes, the evolution of translation machinery followed a gradual addition of scaffold proteins, namely eIF4G, eIF3 and eIF4B, which highly improved the efficiency and regulation of mRNA binding to the 40S ribosomal subunit (*14*). This, together with the incorporation of several RNA helicases, eIF4E and PABP, gradually improved the global efficiency, accuracy and possibilities of gene expression regulation at the level of translation initiation (*14*). Most likely the molecular diversification of the translation apparatus is among the basis that provided to early eukaryotes the scope to invade new ecological niches and overcome the different environmental and biological challenges this represented. Indeed, the evolution of the translation apparatus might have been both, cause and consequence of eukaryotic radiation.

Translation in eukaryotes is tightly coupled to other features and components of cellular metabolism. For example, translation control is coupled to RNA transport to ensure different developmental programs to occur (*107, 108*). The RNA transport machineries have also diverged in different phyla, and together with them some components of the translation apparatus also diverged (*108*). Another fundamental aspect of RNA metabolism is the storage and degradation of mRNAs in different cytoplasmic bodies, such as P-bodies and Pgranules, which contain translation factors (*109*). The diversity and conservation of these foci across phyla are a reflection of the general evolution that the translation machinery and its regulation have undergone during eukaryotes evolution.

#### **Author details**

42 Cell-Free Protein Synthesis

exclusively in fungi.

radiation.

**6. Concluding remarks**

exception of some vascular plants and some ciliates, eukaryotes contain only one *eRF1* gene (*86-89*). Interestingly, unusually high rates of eRF1 evolution have been found in organisms with variant genetic codes (mostly protists and unicellular fungi), especially in the Nterminal domain, which is responsible for stop-codon recognition (*86, 87, 89-92*). eRF1 displays structural similarity to tRNA molecules and mimics its activity during binding of ribosomal A-site during recognition of a stop codon (*91-94*). Since mutations in eRF1 Nterminal domain switch from omnipotent to bipotent mode for stop-codon specificity (*94- 98*), most likely the accelerated evolution of eRF1 in organisms with variations to the nuclear

Another striking case of evolutionary divergence was found in the fungi. Ribosomes from all eukaryotes perform elongation with eEF1A and eEF2. Surprisingly, it was found that the yeast *Saccharomyces cerevisiae* requires an additional essential factor, eEF3, for the elongation cycle to proceed (*100*). Genes encoding eEF3 were subsequently identified exclusively in other fungi, including *Candida*, *Pneumocystis*, *Neurospora*, *Aspergillus* and *Mucor* (*101-104*). eEF3 is an ATPase that interacts with both ribosomal subunits and that is required for the binding of aminoacyl-tRNA-eEF1A-GTP ternary complex to the ribosomal A-site by enhancing the rate of deacylated tRNA dissociation from the E-site (*105*). Recently, it was shown that post-termination complex, consisting of a ribosome, mRNA, and tRNA, is disassembled into single components by ATP and eEF3. Because the release of mRNA and deacylated tRNA and ribosome dissociation takes place simultaneously and no 40S⎯mRNA complexes remain, it is proposed that eEF3 activity promotes ribosome recycling (*106*). It remains unsolved what were the evolutionary forces that led to the emergence of eEF3

One of the enigmas in current Biology is how eukaryotic protein synthesis emerged. I have discussed that, in the absence of SD sequence in mRNAs and RPS1 in ribosomes, the evolution of translation machinery followed a gradual addition of scaffold proteins, namely eIF4G, eIF3 and eIF4B, which highly improved the efficiency and regulation of mRNA binding to the 40S ribosomal subunit (*14*). This, together with the incorporation of several RNA helicases, eIF4E and PABP, gradually improved the global efficiency, accuracy and possibilities of gene expression regulation at the level of translation initiation (*14*). Most likely the molecular diversification of the translation apparatus is among the basis that provided to early eukaryotes the scope to invade new ecological niches and overcome the different environmental and biological challenges this represented. Indeed, the evolution of the translation apparatus might have been both, cause and consequence of eukaryotic

Translation in eukaryotes is tightly coupled to other features and components of cellular metabolism. For example, translation control is coupled to RNA transport to ensure different developmental programs to occur (*107, 108*). The RNA transport machineries have also diverged in different phyla, and together with them some components of the translation

genetic code has been driven mainly to accommodate these variations (*89-99*).

Greco Hernández

*Division of Basic Research, National Institute for Cancer (INCan), Tlalpan, Mexico City, Mexico*

#### **Acknowledgement**

I was supported by the National Institute for Cancer (Instituto Nacional de Cancerología, México).

#### **7. References**

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311-321.

886.

*albicans*. *Yeast 8*, 337-352.

genetic codes. *Nucleic Acids Res. 29*, 921-927.

evolvability of the genetic code. *Nature Rev. Genet. 2*, 49-58.

nuclear genetic code change in ciliates. *Curr. Biol. 11*, 65-74.

[88] Atkinson, G. C., Baldauf, S. L., and Hauryliuk, V. (2008) Evolution of nonstop, no-go and nonsense-mediated mRNA decay and their termination factor-derived components. *BMC Evol. Biol. 8*, 290-308.

48 Cell-Free Protein Synthesis

[74] Keeling, P. J., and Inagaki, Y. (2004) A class of eukaryotic GTPase with a punctate distribution suggesting multiple functional replacements of translation elongation

[75] Sakaguchi, M., Takishita, K., Matsumoto, T., Hashimoto, T., and Inagaki, Y. (2009) Tracing back EFL gene evolution in the cryptomonads-haptophytes assemblage: separate origins of EFL genes in haptophytes, photosynthetic cryptomonads, and

[76] Gile, G. H., Novis, P. M., Cragg, D. S., Zuccarello, G. C., and Keeling, P. J. (2009) The distribution of Elongation Factor-1 Alpha (EF1alpha), Elongation Factor-Like (EFL), and a non-canonical genetic code in the ulvophyceae: discrete genetic characters support a

[77] Cocquyt, E., Verbruggen, H., Leliaert, F., Zechman, F. W., Sabbe, K., and De Clerck, O. (2009) Gain and loss of elongation factor genes in green algae. *BMC Evol. Biol. 9*, 39. [78] Noble, G. P., Rogers, M. B., and Keeling, P. J. (2007) Complex distribution of EFL and

[79] Kamikawa, R., Inagaki, Y., and Sako, Y. (2008) Direct phylogenetic evidence for lateral transfer of elongation factor-like gene. *Proc. Natl. Acad. Sci. U. S. A. 105*, 6965-6969. [80] Kamikawa, R., Yabuki, A., Nakayama, T., Ishida, K., Hashimoto, T., and Inagaki, Y. (2011) Cercozoa comprises both EF1α-containing and EFL-containing members. *Eur. J.*

[81] Kamikawa, R., Sakaguchi, M., Matsumoto, T., Hashimoto, T., and Inagaki, Y. (2010) Rooting for the root of elongation factor-like protein phylogeny. *Mol. Phylogenet. Evol.*

[82] Gile, G. H., Faktorová, D., Castlejohn, C. A., Burger, G., Lang, B. F., Farmer, M. A., Lukes, J., and Keeling, P. J. (2009) Distribution and phylogeny of EFL and EF1alpha in Euglenozoa suggest ancestral co-occurrence followed by differential loss, *PLoS ONE 4*,

[83] Guo, M., Yang, X. L., and Schimmel, P. (2010) New functions of aminoacyl-tRNA

[84] Szymański, M., Deniziak, M., and Barciszewski, J. (2000) The new aspects of aminoacyl-

[85] Fox, P. L., Ray, P. S., Arif, A., and Jia, J. (2007) Noncanonical Functions of AminoacyltRNA Synthetases in Translational Control, in *Translational control in biology and medicine* (Mathews, M. B., Sonenberg, N., and Hershey, J. W. B., Eds.), pp 829-854, Cold Spring

[86] Kim, O. T., Yura, K., Go, N., and Harumoto, T. (2005) Newly sequenced eRF1s from ciliates: the diversity of stop codon usage and the molecular surfaces that are important

[87] Moreira, D., Kervestin, S., Jean-Jean, O., and Philippe, H. (2002) Evolution of eukaryotic translation elongation and termination factors: variations of evolutionary rate and

synthetases beyond translation. *Nature Rev. Mol. Cell Biol. 11*, 668-674.

tRNA synthetases. *Acta Biochim. Polonica 47*, 821-834.

for stop codon interactions. *Gene 346*, 277-286.

genetic code deviations. *Mol. Biol. Evol. 19*, 189-200.

Harbor Laboratory Press, Cold Spring Harbor, New York.

EF1alpha proteins in the green algal lineage, *BMC Evolutionary Biology 7*, 82.

factor 1alpha. *Proc. Natl. Acad. Sci. U. S. A. 101*, 15380-15385.

consistent phylogenetic framework. *J. Eukar. Microbiol. 56*, 367-372.

goniomonads. *Gene 441*, 126-131.

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e5162.

[104] Skogerson, L. (1979) Separation and characterization of yeast elongation factors. *Methods Enzymol. 60*, 676-685.

**Chapter 3**

© 2012 Biyani et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Evolutionary Molecular Engineering**

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52311

**1. Introduction**

**to Efficiently Direct** *in vitro* **Protein Synthesis**

With the completion of human genome project in 2003, the 50th anniversary year of the discovery of the structure of DNA, we entered in the post-genomic era that concentrates on harvesting the fruits hidden in the genomic text. Since then we have witnessed the generation of a tremendous volume of DNA information (*genetic information*). As of September 2011, the Genomes OnLine Database (GOLD, http://www.genomesonline.org) has documented 1914 complete genome projects which comprise 1644 bacterial, 117 archaeal and 153 eukaryal genomes [1]. However, only a fraction of these DNA data are associated with their encoded proteins, i.e., their phenotypes (*functional information*) [2]. Even when a phenotype is associated with the encoding gene, the function of a particular gene cannot be fully understood until it is possible to describe all of the phenotypes that result from the wild-type and mutant forms of that gene. Moreover, unlike a genome that contains a fixed number of genes, the levels of proteins within cells are likely an order of magnitude greater than the number of genes. Therefore, the focus of the scientific community has recently been shifted from gene sequencing to annotation of gene function and regulation through elucidation of protein abundance, expression, post-translational modifications, and proteinprotein interactions. While the pre-genomic era which lasted less than 15 years, the postgenomic era can be expected to last much longer, probably extending over several generations, and thus there is an increasing need for high throughput expression of the genome encoded proteins to profile the entire proteome and get a deeper understanding of protein abundance and reveal novel protein functions. Protein synthesis is therefore a powerful tool for large-scale analysis of proteins for a large-variety of low- and highthroughput applications (see Fig.1) and an essential tool for bridging the gap between genomics and proteomics in the post-genomic era. Noteworthy, the ribosome that catalyses and provide the platform for protein synthesis was in the spotlight recently, as the Nobel

Manish Biyani, Madhu Biyani, Naoto Nemoto and Yuzuru Husimi

## **Evolutionary Molecular Engineering to Efficiently Direct** *in vitro* **Protein Synthesis**

Manish Biyani, Madhu Biyani, Naoto Nemoto and Yuzuru Husimi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52311

## **1. Introduction**

50 Cell-Free Protein Synthesis

*Methods Enzymol. 60*, 676-685.

ATP. *Proc. Natl. Acad. Sci. U. S. A. 107*, 10854-10859.

regulators. *Comp. Funct. Genom.* In press.

*Nature 443*, 663-668.

*Genom.* In press.

[104] Skogerson, L. (1979) Separation and characterization of yeast elongation factors.

[105] Andersen, C. B. F., Becker, T., Blau, M., Anand, M., Halic, M., Balar, B., Mielke, T., Boesen, T., Pedersen, J. S., Spahn, C. M., Kinzy, T. G., Andersen, G. R., and Beckmann, R. (2006) Structure of eEF3 and the mechanism of transfer RNA release from the E-site.

[106] Kurata, S., Nielsen, K. H., Mitchell, S. F., Lorsch, J. R., Kaji, A., and Kaji, H. (2010) Ribosome recycling step in yeast cytoplasmic protein synthesis is catalyzed by eEF3 and

[107] Gamberi, C., and Lasko, P. (2012) The Bic-C family of developmental translational

[108] Vazquez-Pianzola, P., and Suter, B. (2012) Conservation of the RNA transport machineries and their coupling to translation control across eukaryotes. *Comp. Funct.*

[109] Layana, C., Ferrero, P., and Rivera-Pomar, R. (2012) Cytoplasmic ribonucleoprotein foci in eukaryotes: hotspots of bio(chemical)diversity. *Comp. Funct. Genom.* In press.

> With the completion of human genome project in 2003, the 50th anniversary year of the discovery of the structure of DNA, we entered in the post-genomic era that concentrates on harvesting the fruits hidden in the genomic text. Since then we have witnessed the generation of a tremendous volume of DNA information (*genetic information*). As of September 2011, the Genomes OnLine Database (GOLD, http://www.genomesonline.org) has documented 1914 complete genome projects which comprise 1644 bacterial, 117 archaeal and 153 eukaryal genomes [1]. However, only a fraction of these DNA data are associated with their encoded proteins, i.e., their phenotypes (*functional information*) [2]. Even when a phenotype is associated with the encoding gene, the function of a particular gene cannot be fully understood until it is possible to describe all of the phenotypes that result from the wild-type and mutant forms of that gene. Moreover, unlike a genome that contains a fixed number of genes, the levels of proteins within cells are likely an order of magnitude greater than the number of genes. Therefore, the focus of the scientific community has recently been shifted from gene sequencing to annotation of gene function and regulation through elucidation of protein abundance, expression, post-translational modifications, and proteinprotein interactions. While the pre-genomic era which lasted less than 15 years, the postgenomic era can be expected to last much longer, probably extending over several generations, and thus there is an increasing need for high throughput expression of the genome encoded proteins to profile the entire proteome and get a deeper understanding of protein abundance and reveal novel protein functions. Protein synthesis is therefore a powerful tool for large-scale analysis of proteins for a large-variety of low- and highthroughput applications (see Fig.1) and an essential tool for bridging the gap between genomics and proteomics in the post-genomic era. Noteworthy, the ribosome that catalyses and provide the platform for protein synthesis was in the spotlight recently, as the Nobel

© 2012 Biyani et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Prize in Chemistry 2009 was awarded to the work that unlocked the structure and function of the ribosomes.

Evolutionary Molecular Engineering to Efficiently Direct *in vitro* Protein Synthesis 53

drawback of synthesizing proteins in the lysate is that the lysate contains a large portion of the cellular proteins and nucleic acids that are not necessarily involved in the targeted protein synthesis and can lead to low protein yields through interfering with the subsequent purification reactions. In addition, the presence of proteases and nucleases in the lysates could be inhibitory to protein synthesis. In order to addressing this issue, cell-free protein synthesis system was reconstituted *in vitro* from purified components of the *E. coli* translation machinery. This system, termed the "protein synthesis using recombinant elements" (PURE) system, contains all necessary translation factors, purified with high specific activity, and allows efficient protein production [12]. Remarkably, this reconstituted system has been shown to catalyze efficient in vitro protein synthesis by providing a much

The second issue is that existing cell-free systems differ substantially from each other with respect to their efficiency and scalability to produce proteins and therefore these systems has to be programmed for given exogenous mRNA templates. Although different lysates may contain specific cellular factors that promote protein synthesis, a key factor in ensuring high protein production is the use of strong translational enhancer sequences (untranslated regions, UTRs) in the mRNA templates, which has long been known to enhance protein production up to several hundred-folds [14]. UTRs are known to play crucial role in the post-transcriptional regulation of gene expression, including modulation of the transport of mRNAs out of the nucleus and of translation efficiency [15]. The average length of UTRs motifs located at the 5'end of the exon, called 5'-UTR, ranges between 100 and 200 nucleotides and strikingly varies a lot within a species, e.g., in humans, the longest known 5'UTR is 2,803 while the smallest is just 18 nucleotides

**Figure 2.** The average length of untranslated region sequences in the different taxonomic classes. Grey

(coldblooded) Rodent Human

Fungus Plant Invertebrate Vertebrate

**Complexity**

The structural features of the 5'UTR have a major role in the control of protein synthesis. Those proteins which are involved in developmental processes, including growth factors, transcription factors or proto-oncogenes, often have longer 5'UTR than an average and thus

cleaner background than a lysate-based system [13].

bar representing 5'-UTR and black bar is 3'-UTR.

5'UTR 3'UTR

[16,17] (Fig.2).

Mean UTR length (nt)

## **2. Significance of protein synthesis enhancer sequences (5'UTR)**

Cell-based (*in vivo*) and cell-free (*in vitro*) methods have been developed for production of protein synthesis [3]. Cell-based host systems such as bacteria, yeast, worms, mammalians used for protein synthesis and protein expression analysis, however, have been unable to meet the requirement of producing large amounts of purified and functional proteins which is a prerequisite to facilitate structure-based functional analysis. For example, purified proteins are necessary to grow protein crystals whose X-ray diffraction patterns provide the most precise structural information. Other limitations in host organisms includes such as bacteria don't have the intracellular organelles found in eukaryotes; yeast lack a dimension of complexity in intracellular communication observed in metazoans; and even other mammalian system are different from human in important aspects of both normal physiology and disease pathogenesis [4]. In addition, many biochemical pathways are simply difficult to study in the larger context of other events happening at the same time within the cell. In contrast to the cell-based systems, cell-free protein expression systems are now becoming the favored alternative with far greater fidelity as it offers a simple and flexible system for the rapid synthesis of functional proteins. There is currently a wide range of cell-free translation systems due to the ready availability of cell extracts prepared from various cell sources, including *Escherichia coli*, yeast, wheat germ, rabbit reticulocytes, *Drosophila* embryos, hybridomas, and insect, mammalian, and human cells [5-11]. Although encouraging, there would be some major issues in the use of cell-free systems. First, a major

**Figure 1.** Application of Protein synthesis

drawback of synthesizing proteins in the lysate is that the lysate contains a large portion of the cellular proteins and nucleic acids that are not necessarily involved in the targeted protein synthesis and can lead to low protein yields through interfering with the subsequent purification reactions. In addition, the presence of proteases and nucleases in the lysates could be inhibitory to protein synthesis. In order to addressing this issue, cell-free protein synthesis system was reconstituted *in vitro* from purified components of the *E. coli* translation machinery. This system, termed the "protein synthesis using recombinant elements" (PURE) system, contains all necessary translation factors, purified with high specific activity, and allows efficient protein production [12]. Remarkably, this reconstituted system has been shown to catalyze efficient in vitro protein synthesis by providing a much cleaner background than a lysate-based system [13].

52 Cell-Free Protein Synthesis

of the ribosomes.

**Figure 1.** Application of Protein synthesis

Prize in Chemistry 2009 was awarded to the work that unlocked the structure and function

Cell-based (*in vivo*) and cell-free (*in vitro*) methods have been developed for production of protein synthesis [3]. Cell-based host systems such as bacteria, yeast, worms, mammalians used for protein synthesis and protein expression analysis, however, have been unable to meet the requirement of producing large amounts of purified and functional proteins which is a prerequisite to facilitate structure-based functional analysis. For example, purified proteins are necessary to grow protein crystals whose X-ray diffraction patterns provide the most precise structural information. Other limitations in host organisms includes such as bacteria don't have the intracellular organelles found in eukaryotes; yeast lack a dimension of complexity in intracellular communication observed in metazoans; and even other mammalian system are different from human in important aspects of both normal physiology and disease pathogenesis [4]. In addition, many biochemical pathways are simply difficult to study in the larger context of other events happening at the same time within the cell. In contrast to the cell-based systems, cell-free protein expression systems are now becoming the favored alternative with far greater fidelity as it offers a simple and flexible system for the rapid synthesis of functional proteins. There is currently a wide range of cell-free translation systems due to the ready availability of cell extracts prepared from various cell sources, including *Escherichia coli*, yeast, wheat germ, rabbit reticulocytes, *Drosophila* embryos, hybridomas, and insect, mammalian, and human cells [5-11]. Although encouraging, there would be some major issues in the use of cell-free systems. First, a major

**2. Significance of protein synthesis enhancer sequences (5'UTR)**

The second issue is that existing cell-free systems differ substantially from each other with respect to their efficiency and scalability to produce proteins and therefore these systems has to be programmed for given exogenous mRNA templates. Although different lysates may contain specific cellular factors that promote protein synthesis, a key factor in ensuring high protein production is the use of strong translational enhancer sequences (untranslated regions, UTRs) in the mRNA templates, which has long been known to enhance protein production up to several hundred-folds [14]. UTRs are known to play crucial role in the post-transcriptional regulation of gene expression, including modulation of the transport of mRNAs out of the nucleus and of translation efficiency [15]. The average length of UTRs motifs located at the 5'end of the exon, called 5'-UTR, ranges between 100 and 200 nucleotides and strikingly varies a lot within a species, e.g., in humans, the longest known 5'UTR is 2,803 while the smallest is just 18 nucleotides [16,17] (Fig.2).

**Figure 2.** The average length of untranslated region sequences in the different taxonomic classes. Grey bar representing 5'-UTR and black bar is 3'-UTR.

The structural features of the 5'UTR have a major role in the control of protein synthesis. Those proteins which are involved in developmental processes, including growth factors, transcription factors or proto-oncogenes, often have longer 5'UTR than an average and thus untranslated regions of mRNAs have crucial roles in protein regulation through protein synthesis. Structural elements of the eukaryotic mRNA, including the 5'cap and 3'poly(A) tail, and a series of protein-mRNA and protein-protein interactions, including several eIF (eukaryotic initiation factors), are important determinants of translation initiation (Fig.3). In eukaryotes, a multifactor complex of eukaryotic initiation factors are involved in the initiation phase of protein synthesis. But, in particular, 5'UTR plays a major role in the translation initiation, a critical step in protein synthesis which is determining qualitatively and quantitatively which proteins are made, when and where. 5'UTR is composed of several regulatory elements, including the Shine-Dalgarno (SD) and the AU-rich sequences which facilitates 16s rRNA-specific ribosome binding to initiate the protein synthesis [18,19]. In cell-based or in vivo systems, the translation of natural mRNAs is finely regulated by several mechanisms using 5'-capped and 3'-poly(A) containing long-untranslated regions (UTRs).

Evolutionary Molecular Engineering to Efficiently Direct *in vitro* Protein Synthesis 55

Therefore, it is prerequisite to find an alternate for natural UTRs dependency and optimization of translation initiation in cell-free system for next-generation in vitro high throughput protein synthesis systems. In a recent study using cell-free systems, the translation-enhancing activity of some commonly used natural enhancer sequences, such as omega from tobacco mosaic virus and the 5'UTR of β-globin mRNA from *Xenopus laevis*, was reported to vary from 1- to 10-fold, depending on the source of the cell-free extract used (e.g., wheat germ, rabbit reticulocyte lysate, insect) [20]. Therefore, optimization of enhancer sequences of an exogenous mRNA template with a given crude cell extract is desirable before using a cell-free protein synthesis system. A recent new development has been the remarkable generation of a universal cell-free translation system that mediates efficient translation in multiple prokaryotic and eukaryotic systems by bypassing the need for early

**3. Co-evolutionary relationship between translational initiation and**

In the course of evolution on the Earth, how the early life evolved beginning with a hypothetical RNA world-to-the world we know today (DNA world) is the persistent issue of debate for evolutionary biologist. In 1968, Francis Crick argued about the existence of the RNA world in the initial stage of evolution in which RNA molecules assembled from a nucleotide soup and supposed to carry both the genetic and catalytic information (Fig.4). In later stage, some special types of RNA molecules (now termed as Ribozymes) was considered to catalyzes its own self-replication and therefore to develop an entire range of enzymatic activities to form DNA world through an intermediate RNP (RNA/Protein) world. However, there are certain questions that cannot be answered with proposed RNP world. These include: 1. How did 'RNA-world (Ribozyme-type)' evolved to 'DNA-world (cell-type)' since there is no record exists of the intermediates between the RNA-world and organized complexity of cell? 2. What was the first Protein evolved out of an RNA world? 3. How could it have evolved and how the process of translation emerged? 4. If ribosome make protein then how the first ribosomal protein appeared? 5. Why is ribosome made half

The recent advances in evolutionary molecular engineering have revealed the bonding strategy of the genotype to its phenotype as a unique and essential nature of a 'virus' and thus the role of virus-type strategy in the course of evolution on the Earth. In 1995, Nemoto and Husimi proposed a 'virus-early and cell-late model' that a virus-like molecule consist of genotype (mRNA) and phenotype (its coded protein) molecules emerged in the latter period of RNA world was the key molecule which enforced the transition from RNA-to-RNP world by co-evolving the translation system and a virus-like molecule coded a primitive protein of replicase [22]. In this theory, they also showed that such virus-like molecule could introduce Darwinian evolution into the Eigen's hypercycle members (RNA replicase of RNA, RNA translation members, RNA replicase of protein) resulting in carrying out co-evolution

translation initiation steps [21].

**protein synthesis**

of protein and half of RNA ?

**Figure 3.** A schematic drawing of a eukaryotic mRNA, illustrating some post-transcriptional regulatory elements that affect initiation of protein synthesis.

Therefore, the efficiency of a cell-free translation system which is reconstituted using crude cell extract is restricted due to the problematic of maintaining long-natural UTRs in the in vitro construct. Even if so, the obvious question here is that "are the natural UTRs can meet the requirements of various translation factors in a cell-free system to carry similar mechanisms as in in vivo system?" Looking at this 'black box' may open a new window into the post-transcriptional regulation of gene expression using cell-free translation systems. Therefore, it is prerequisite to find an alternate for natural UTRs dependency and optimization of translation initiation in cell-free system for next-generation in vitro high throughput protein synthesis systems. In a recent study using cell-free systems, the translation-enhancing activity of some commonly used natural enhancer sequences, such as omega from tobacco mosaic virus and the 5'UTR of β-globin mRNA from *Xenopus laevis*, was reported to vary from 1- to 10-fold, depending on the source of the cell-free extract used (e.g., wheat germ, rabbit reticulocyte lysate, insect) [20]. Therefore, optimization of enhancer sequences of an exogenous mRNA template with a given crude cell extract is desirable before using a cell-free protein synthesis system. A recent new development has been the remarkable generation of a universal cell-free translation system that mediates efficient translation in multiple prokaryotic and eukaryotic systems by bypassing the need for early translation initiation steps [21].

54 Cell-Free Protein Synthesis

(UTRs).

**5'UTR**

untranslated regions of mRNAs have crucial roles in protein regulation through protein synthesis. Structural elements of the eukaryotic mRNA, including the 5'cap and 3'poly(A) tail, and a series of protein-mRNA and protein-protein interactions, including several eIF (eukaryotic initiation factors), are important determinants of translation initiation (Fig.3). In eukaryotes, a multifactor complex of eukaryotic initiation factors are involved in the initiation phase of protein synthesis. But, in particular, 5'UTR plays a major role in the translation initiation, a critical step in protein synthesis which is determining qualitatively and quantitatively which proteins are made, when and where. 5'UTR is composed of several regulatory elements, including the Shine-Dalgarno (SD) and the AU-rich sequences which facilitates 16s rRNA-specific ribosome binding to initiate the protein synthesis [18,19]. In cell-based or in vivo systems, the translation of natural mRNAs is finely regulated by several mechanisms using 5'-capped and 3'-poly(A) containing long-untranslated regions

**Cap**

**eIF4E**

**eIF4A**

**eIF1A**

5' 3'

**eIF4G**

**3'UTR**

**Figure 3.** A schematic drawing of a eukaryotic mRNA, illustrating some post-transcriptional regulatory

**ORF**

**40S**

**eIF3 complex**

**eIF4B**

Therefore, the efficiency of a cell-free translation system which is reconstituted using crude cell extract is restricted due to the problematic of maintaining long-natural UTRs in the in vitro construct. Even if so, the obvious question here is that "are the natural UTRs can meet the requirements of various translation factors in a cell-free system to carry similar mechanisms as in in vivo system?" Looking at this 'black box' may open a new window into the post-transcriptional regulation of gene expression using cell-free translation systems.

elements that affect initiation of protein synthesis.

**eIF5**

**eIF1**

**eIF2 GTP Met**

## **3. Co-evolutionary relationship between translational initiation and protein synthesis**

In the course of evolution on the Earth, how the early life evolved beginning with a hypothetical RNA world-to-the world we know today (DNA world) is the persistent issue of debate for evolutionary biologist. In 1968, Francis Crick argued about the existence of the RNA world in the initial stage of evolution in which RNA molecules assembled from a nucleotide soup and supposed to carry both the genetic and catalytic information (Fig.4). In later stage, some special types of RNA molecules (now termed as Ribozymes) was considered to catalyzes its own self-replication and therefore to develop an entire range of enzymatic activities to form DNA world through an intermediate RNP (RNA/Protein) world. However, there are certain questions that cannot be answered with proposed RNP world. These include: 1. How did 'RNA-world (Ribozyme-type)' evolved to 'DNA-world (cell-type)' since there is no record exists of the intermediates between the RNA-world and organized complexity of cell? 2. What was the first Protein evolved out of an RNA world? 3. How could it have evolved and how the process of translation emerged? 4. If ribosome make protein then how the first ribosomal protein appeared? 5. Why is ribosome made half of protein and half of RNA ?

The recent advances in evolutionary molecular engineering have revealed the bonding strategy of the genotype to its phenotype as a unique and essential nature of a 'virus' and thus the role of virus-type strategy in the course of evolution on the Earth. In 1995, Nemoto and Husimi proposed a 'virus-early and cell-late model' that a virus-like molecule consist of genotype (mRNA) and phenotype (its coded protein) molecules emerged in the latter period of RNA world was the key molecule which enforced the transition from RNA-to-RNP world by co-evolving the translation system and a virus-like molecule coded a primitive protein of replicase [22]. In this theory, they also showed that such virus-like molecule could introduce Darwinian evolution into the Eigen's hypercycle members (RNA replicase of RNA, RNA translation members, RNA replicase of protein) resulting in carrying out co-evolution between translation system and protein replicase. This was later reinforced by inventing and demonstrating a genotype-phenotype linked method (IVV, in vitro virus) for evolutionary molecular engineering [26] and this strongly suggest the potential of IVV method to understand the relation between ribosome-mRNA interaction.