the first glimpse of the ctd structure was through its interaction with human pin1 , a unique prolyl isomerase that catalyzes cis / trans isomerization of specific phos.ser/thr-pro motifs in signaling proteins ( 1316 ) .
identification and characterization of this novel peptidyl - prolyl cis / trans isomerase ( ppiase ) , pin1 , led to discovery of a novel postphosphorylation regulatory mechanism , in which regulation is achieved by conformational changes of a phosphorylated ser / thr - pro peptide bond upon proline isomerization ( fig .
this change in the configuration of the polypeptide has a profound effect on pin1 targets and therefore modulates various signaling pathways at both transcriptional and posttranslational levels .
specifically , activity of prolyl - isomerization of pin1 can interconvert the cis / trans conformation of the phos.ser/thr-pro motif of target proteins and make them better or worse substrates for conformation - specific signaling kinases ( such as cyclin - dependent protein kinase , glycogen synthetase kinase 3  , and mitogen - activated protein kinase ) and phosphatases ( such as pp2a and cdc25 ) .
recent studies also provide substantial evidence implicating pin1 in progression of malignant tumor cells ( 17 ) and development of alzheimer 's disease ( 18 ) .
high affinity inhibitory unnatural peptides have been developed as a good template for chemical compounds targeting pin1 for antineoplastic effects ( 19 ) .
the yeast homologue of pin1 , ess1 , interacts physiologically and genetically with the ctd ( 20 ) .
furthermore , hyperphosphorylated rna polymerase ii appears to be the dominant binding target in yeast extracts ( 21 ) .
considering the high local concentration of the phos.ser/thr-pro motif in the hyperphosphorylated ctd , it is plausible that the ctd is the major substrate of pin1 in vivo .
the binding of a single ctd repeat that is phosphorylated at ser5 was reported to be 30 m ( 22 ) .
presumably , the ctd tail containing 2652 such repeats localizes a substantial amount of pin1 .
pin1 is a 163 amino acid polypeptide that can be divided into two domains based on topology and function , a c - terminal ppiase domain and an n - terminal ww domain .
structure of the ww domain reveals three antiparallel -strands forming a shallow interface with the ppiase domain for substrate peptide binding ( 23 , 24 ) .
it has long been realized that ww domains recognize proline - containing sequences but it was not clear until recently that ww domains join a group of modules that bind to protein ligands in a phosphorylation - dependent manner ( 25 , 26 ) ; these domains include sh2 , ptb , 14 - 3 - 3 , wd40 , fha , and ff domains .
the modular nature of ww domain interactions leads to a classification into four distinct groups based on binding specificity ( 27 ) .
group i ww domains , such as dystrophin and the yes - associated protein yap65 , recognize 
group ii , such as fe65 and forming binding proteins ( fbps ) , bind the 
group iii ww domains select polyproline motifs flanked by arginine or lysine ( 31 , 32 ) .
group iv ww domain , including human pin1 and nedd4 , specifically recognize a phos.ser/thr-pro motif ( 22 , 26 , 33 ) . the molecular detail of recognition of the ctd by pin1 was elucidated by a structure of human pin1 in complex with one doubly phosphorylated ctd repeat ( 22 ) ( fig .
so far , this is the only structure available for a full - length pin1 binding to its target at the recognition module ww domain .
the structure is consistent with thermodynamic data , which the phosphorylated ser5 in the ctd repeat is the major binding element in recognition .
loop 1 of the ww domain has been shown to be highly flexible in apo pin1 ( 34 ) but this loop is essential for specificity recognition ( 35 ) . in the complex structure , this loop warps toward the substrate peptide to ensure binding and results in an exaggerated twist in the triple - stranded -sheet ( 22 ) ( fig .
this twist is coupled to a contraction of the ww domain ligand binding surface formed between the ww and ppiase domains .
the two essential elements for peptide recognition include binding of phosphate by loop 1 residues and hydrophobic stacking of proline by tyr23 and trp34 ( 22 ) .
the binding of pin1 to the ctd can modulate the regulatory effect of other ctd - binding proteins . in vitro experiments
showed that pin1 can influence the phosphorylation status of the ctd by inhibiting the transcription factor iif - interacting ctd phosphatase 1 ( fcp1 ) and stimulating ctd phosphorylation by cdc2/cyclinb ( 36 , 37 ) .
the rsp5 , a ubiquitin ligase that binds to the ctd , also functions to oppose pin1 effects on rna polymerase ii ( 38 ) .
the biological and structural results of pin1 effects on rna polymerase ii function support a model that pin1 works in a processive manner on the ctd with the ww domain acting as a binding element restricting movement to an efficient one - dimensional walk and with the ppiase acting much like a reading head to processively isomerize the peptide bonds .
the binding of pin1 prolongs the phosphorylated state of the ctd by suppressing dephosphorylation , thereby enhancing the regulatory effect of ctd binding proteins . ribbon representation of four ctd recognition modules . ( a ) ww domain of pin1 in complex with a short ctd peptide ( 1f8a ) ; ( b ) cid domain of pcf11 in complex with a short ctd peptide ( 1sza ) ; ( c ) sri domain of set2 ( 2a7o ) ; and ( d ) ff domain of ca150 ( 2kis ) .
recognition of the phosphorylated ctd by pin1 is mediated by its ww domain , a modular domain of around 40 residues that is essential for recognition of proline - rich motifs by the ppiase domain .
a more specific recognition domain for the ctd is the ctd - interacting domain ( cid ) identified in multiple rna processing and termination factors in eukaryotes ( 39 , 40 ) ( fig .
4 ) . superimposition of cid domains from pcf11 ( light blue , 1sza ) , nrd1 ( light pink , 3clj ) , and scaf8 ( white , 3d9i ) . in yeast ,
the pcf11 is a yeast protein of 70 kd with a cid domain directly targeted to the ctd of rna polymerase ii , which gives us the first glance of how a cid recognizes heptad repeats of the ctd .
interestingly , the cid domain of pcf11 can bind to both unphosphorylated or phos.ser2 ctd in biophysical binding assays ( 39 ) .
consistent with the biochemical data for such preferences , the co - crystal structure of the pcf11 cid and a ctd peptide shows no direct interaction between the phosphate group of phos.ser2 and pcf11 , indicating no prerequirement of phosphorylation of the ctd for protein binding ( 39 ) ( fig .
3b ) . the cid domain , as an eight - helical bundle , recognizes a span of two heptad repeats in the ctd with a conserved groove , whereas the phosphate group of phos.ser2 actually forms an intramolecular hydrogen bond with thr4 of the ctd and stabilizes the sharp -turn formed by ctd , presenting the side chain to the binding groove ( fig .
hydrogen bondings between the cid domain and the ctd peptide are distributed between the cid side chain and the main chain amide and carbonyl group of the ctd peptide .
importantly , hydrophobic interaction by tyr1 of the ctd to cid might contribute greatly for the specificity for phos.ser2 over phos.ser5 .
meinhart and cramer ( 39 ) conclude that ser2 phosphorylation stabilizes the ctd -spiral that is incorporated into the transcription complex .
ser5 phosphorylation would unwind the spiral resulting in an extended region that binds the capping enzyme .
overall , the binding of pcf11 is not particularly strong but is consistent with its dynamic role involved in dismantling the elongation complex ( 41 , 42 ) .
another cid containing protein nrd1 is an essential player in the termination pathway for rna polymerase ii - mediated transcription for snorna , snrna as well as cryptic unstable transcripts ( cut ) .
the complex of nrd1-nab3-sen1 is recruited to the transcription machinery through the ctd by the cid domain of nrd1 of the complex .
4 ) has a very similar overall fold and a strong conservation of residues involved in ctd peptide binding with pcf11 , but nrd1 shows a different specificity profile in which a much stronger preference for phos.ser5 over phos.ser2 in the ctd sequence is detected for the cid from nrd1 by in vitro binding assays ( 43 ) . using yeast - two hybrid and fluorescence anisotropy , vasiljeva showed a tenfold improvement in binding affinity for singly phosphorylated ctd double repeats at ser5 over ser2 ( kd=40 m for phos.ser5 vs. 390 m for phos.ser2 ) and a slight improvement when the peptide is doubly phosphorylated ( kd=16 m upon double phosphorylation for both ser2 and ser5 sites ) .
specificity for phos.ser5 on the protein is essential to understand how different termination pathways are selected .
a potential different phosphate binding site was proposed but more insightful information about specificity will only be available with a structure of the nrd1cid complexed with the ctd peptide . since the nrd1-dependent termination pathway is usually associated with much shorter transcripts ( a few hundred base - pairs ) , upon which point dephosphorylation of ser5
is not complete , a logical hypothesis is that phosphorylation at ser5 favors the selection of nrd1 complex as a termination pathway in yeast . indeed , gudipati et al .
( 44 ) showed that reducing phosphorylation of ser5 using a mutant of kin28 , a ctd ser5 kinase , will hamper nrd - dependent termination .
the selectivity for phos.ser5 over phos.ser2 might be the determining factor for the selection of termination pathways . a functional model for how the phosphorylation pattern of the ctd determines the transcriptional pathways suggests that termination by the nrd1-nab3-sen1 complex is enforced when the ctd is still highly phosphorylated at ser5 ( 45 ) .
the structure of a complex of the cid of nrd1 with the ctd domain will help provide a molecular explanation of transcription termination .
another cid containing protein human scaf8 was crystallized with different phosphorylated forms of ctd peptides ( fig .
4 ) , providing more clues about how registration of a phosphate group on the ctd is encoded in target recognition for cid domains ( 46 ) .
the scaf8 is implicated in splicing with a preference of phos.ser2 , similar to mrna 3-processing factor pcf11 .
indeed , a similar conformation of beta - turn adapted by the ctd is observed in scaf8 upon peptide binding but with one major distinction : phos.ser2 is directly recognized by a basic residue , arg112 , through salt bridge interaction ( 46 ) . at a similar position in pcf11
the replacement of methionine by arginine in scaf8 distinguishes the phos.ser2 ctd from the unphosphorylated form and might explain a tighter interaction for scaf8 toward phosphoryl - peptide ( 68 m ) . in the study of becker et al .
( 46 ) , an issue was raised whether phosphorylated ser7 contributes to recognition by the cid domain of scaf8 .
binding affinity measured by fluorescence anisotropy showed no advantage with additional ser7 phosphorylation ( 68 m for phos.ser2 and 90 m for doubly phosphorylated ser2 and ser7 ) .
consistent with the binding interaction measurement , the structure of the complex with the scaf8 cid presents no interaction between the phosphate group of ser7 with the protein .
a wide range of enzymes participate in dynamic modifications of the ctd , including kinases and phosphatases responsible for addition and removal of phosphates . the ctd is principally phosphorylated by cyclin - dependent kinases ( cdks ) with their associated cyclins .
specifically , ser5 phosphorylation is mainly catalyzed by cdk7/cyclin h subunits of tfiih ( 4749 ) ; ser2 phosphorylation is mainly catalyzed by p - tefb , which contains cdk9/cyclin t subunits ( 50 , 51 ) .
intriguingly , it is suggested that cdk9 also makes a contribution to ser5 phosphorylation and the relative contribution of tfiih - associated cdk7 varies between different genes based on experimental observations ( 52 ) .
moreover , the ctd can also be phosphorylated at both ser2 and ser5 by cdk8 as part of the mediator complex , and preferentially phosphorylated at ser5 by mapk2/erk2 ( 53 ) . recently
, tfiih - associated cdk7 kinase has also been shown to phosphorylate ser7 in vivo ( 52 , 54 ) . even though structural information has been obtained for cdk7 ( 55 ) and cdk9/cycln t ( 56 ) ,
how they recognize the ctd peptide and label the phosphorylation mark on ctd is still elusive .
it is assumed that specificity is achieved by other associated proteins in the multiprotein complexes they are involved ( cdk7 in tfiih , cdk8 in mediator , and cdk9 in p - tefb ) .
dephosphorylation is essential for recycling rna polymerase ii , because after each round of transcription , the ctd has to be dephosphorylated in order to actively restart a new round of transcription . in humans ,
fcp1 , which is required for general transcription and cell viability , was the first discovered ctd - specific phosphatase with a catalytic preference for phos.ser2 .
the fcp1 is conserved among eukaryotes and was shown to be essential for cell survival in budding and fission yeast ( 57 , 58 ) .
the conserved region of fcp1 is composed of two domains : an n - terminal fcp homology ( fcph ) domain with phosphatase activity and a c - terminal breast cancer protein related c - terminal ( brct ) domain ( 59 ) ( fig . 5 ) .
the zoom - in picture shows the pro3 binds to the hydrophobic pocket with the aromatic residues shown in stick .
the active site signature motif is colored with pale green , and the insertion domain is colored with light pink .
recently , a family of small ctd phosphatases ( scps ) with activities preferential for phos.ser5 was identified ( 60 , 61 ) .
the scps also contain the fcph catalytic domain that includes the dxdx(t / v ) motif , the signature of a superfamily of phosphotransferases and phosphohydrolases called the haloacid dehalogenase ( had ) superfamily ( 62 ) .
interestingly , outside the signature motif , scps share very little sequence similarity with the other enzymes in the had superfamily ( 63 ) . in humans
, scp1 has more than 20% sequence identity to fcp1 in the fcph domain but lacks the c - terminal brct domain that exists in fcp1 . moreover , scp2 and scp3 , which also lack the brct domain , share more than 90% similarity with scp1 in the fcph domain ( 60 ) ( fig .
( 64 ) showed a central parallel  sheet flanked by two  helices , a two - stranded  sheet , and a short 310 helix .
the conserved dxdx(t / v ) signature motif lines part of a central crevice , which forms the active site and coordinates the mg ion that is essential to scp1 phosphatase activity .
the first aspartate in the signature motif is involved in mg - assisted phosphoryl transfer and acts as the phosphoryl acceptor .
mutation of this residue ( asp96 in scp1 ) to alanine or asparagine abolished the activity of scp1 .
the second aspartate ( asp98 in scp1 ) also contributes to metal ion - binding and could possibly function as a general acid / base ( 64 ) .
the proposed phosphoryl transfer mechanism for the scp / fcp family involves a phosphoryl - aspartate intermediate .
existence of this phosphoryl - enzyme intermediate was confirmed in a recent structural and functional study when we successfully trapped the phosphoryl - aspartate intermediate in the crystal structure of an scp1d206a mutant soaked with para - nitrophenyl phosphate ( pnpp ) ( 63 ) .
the steady - state kinetic analysis of a variety of scp1 mutants revealed the importance of asp206 in mg coordination mediated by a water molecule .
moreover , snapshots of the phosphoryl transfer reaction at each stage of scp1-mediated catalysis were also captured in this study . in order to understand the discrimination of phos.ser5 over phos.ser2 as a substrate by scp1 , the complex structure of a dominant negative form of human scp1 ( scp1d96n ) bound with ser2/ser5-phosphorylated ctd peptide
the defined complex structure revealed a unique binding mode of the peptide in which ser2pro3thr4(phos.ser5 ) forms a -turn .
the pro3 is recognized by an aromatic - rich hydrophobic pocket near the active site that further confers substrate specificity ( 65 ) .
notably , scp1 shows remarkable specificity toward the trans peptide - bond configuration of the two prolines in the ctd repeat , which can adopt both cis and trans configurations .
such configuration switching is known to modulate the structure of the ctd and its accessibility to kinases or phosphatases ( 21 , 22 ) .
an insertion domain formed by a three - stranded  sheet directly follows the signature motif ( fig .
this insertion domain is unique to fcp1/scp1 family phosphatases and may assist in substrate recognition .
consistent with the known specificity of scp1 toward phos.ser5 instead of phos.ser2 , in the complex structure the phos.ser2 flips out of the active site , making no direct interaction with the protein .
even though scp and fcp share similar phosphatase active sites , their strategy for substrate recognition might be different , as suggested by the recent crystal structure of apo schizosaccharomyces pombe fcp1 ( spfcp1 ) ( 59 ) ( fig .
the minimal effective ctd substrate for spfcp1 is a single heptad ctd peptide : ser5pro6ser7tyr1(phos.ser2)pro3thr4 , among which the tyr1 and pro3 flanking the phos.ser2 are critical determinants of fcp1 activity ( 66 ) .
the spfcp1 structure revealed that it is a y - shaped protein composed of three structural domains ( 59 ) .
the stem of the y is the fcph domain that contains a globular catalytic phosphatase core similar to that of scp1 .
one major difference is that the three - stranded  sheet in scp1 ( insertion domain ) is accessible for substrate recognition , whereas in fcp1 it is buried by a helical insertion domain , suggesting a different binding interface between fcp and the ctd with phos.ser2 ( fig .
even though 2652 heptad repeats exist in the ctd primary sequence , recognition of the ctd by proteins in all the complex structures discussed above only show a spanning of one or two repeats .
this is understandable since a balance between a favorable interaction of the ctd with its binding partners versus the entropy cost for binding a disordered peptide needs to be achieved .
this leads to the proposed mechanism that a double repeat of the ctd sequence is the functional unit for transcription . to explore
if such rule is consistent with the mrna capping enzyme cgt1 , four heptad repeats each with their ser5 phosphorylated were used in a cocrystallization experiment ( 67 ) .
interestingly , a long span of ctd peptide consisting of 17 amino acids was modeled in the density of one of the two monomers with an extensive buried surface of 1,600  ( 67 ) .
three of the four phosphorylated ser5 residues were visible in this structure with both the first and third phosphate group recognized by positively charged patches on the cgt1 surface .
consistent with a previous study of cgt1 , the tyr1 position in the ctd sequence is essential for recognition by the protein ( 68 ) . on the other hand , a single mutation of the cgt protein for
the recognition interface did not show obvious deleterious effects during yeast mutation screening ( 67 ) , possibly due to the extended binding surface without one interaction dominating binding .
since the other monomer in the asymmetric unit shows a much smaller interface , it suggests that multiple phosphorylation sites are not a prerequisite for ligand binding .
further affinity measurements with a different length and registration of the phos.ser5 would elucidate whether interaction with multiple repeats of the ctd is essential for the effective binding by cgt1 .
the identification of the histone methyltransferase set2 as a novel ctd binding partner bridges the ctd code to the histone code by implicating the regulatory effect of the ctd on histone modification and epigenetic control ( 69 , 70 ) ( fig .
the nucleosome is composed of a histone octamer consisting of two copies of each of the core histones h2a , h2b , h3 , and h4 , around which 147 bp of dna is wrapped .
such a structure is not only a strategy to compress large genomic dna , but also provides a potential solution for tight regulation of dna replication , repair , and transcription . during transcription ,
for example , the binding sites for a variety of transcription factors can be occluded by histones , leading to transcriptional silencing ( 71 ) .
access to specific loci on the nucleosomal dna is dynamically regulated by many factors including chromatin modifiers and chromatin remodelers .
histones can be covalently modified by chromatin modifiers at particular loci , most of which are concentrated in the relatively unstructured n - terminal tails of histones .
these modifications include acetylation , methylation , phosphorylation , ubiquitination , and sumoylation ( 72 ) .
the histone code , which is defined by covalent modifications , represents a fundamental regulatory mechanism of gene expression and repression ( 73 ) .
furthermore , the histone code can be interpreted by different modules in a modification - dependent manner to decide whether a gene is to be transcribed .
a novel rpb1-binding domain of set2 , called set2rpb1 interacting ( sri ) domain , mediates the recognition and interaction with the phosphoryl ctd ( fig .
lys4 of histone 3 is methylated by the proteins of the set1 family , while lys36 is methylated by proteins of the set2 family ( 69 ) .
both set1 and set2 associate with the ctd but at different stages of transcription : set1 binds to phosphorylated ctd enriched with ser5 at the promoter region through the mediation of the paf complex ( 74 , 75 ) ; whereas the recognition of the ctd by set2 relies on the phosphorylation of ser2 in heptad repeats ( 76 ) . the sri domains from both human ( 77 ) and yeast ( 78 ) set2 were determined by nmr and the binding interface mapped by phosphoryl - peptide titration .
the study of affinity with different length and phosphorylation registration showed that single repeats are not sufficient for sri recognition and a strong preference for doubly phosphorylated peptide was observed .
five residues were identified as essential for the effective association between set2 sri and the ctd using nmr and mutagenesis ( 74 ) .
one can assume the positively charged residues , lys54 , arg58 , and his62 of the set2 sri , are involved in phosphate binding whereas val31 and phe53 are important for hydrophobic interaction with the side chain residues of the ctd , possibly tyrosine or proline .
in addition , the ff domain is a protein - protein interaction module existing in several ctd interaction proteins such as human transcription factor ca150 ( 79 ) and splicing factor prp40 ( 80 ) . like the sri domain ,
the ff domains usually are arranged as tandem arrays in proteins and such architecture might account for interaction with the ctd with each module contributing very weakly to binding .
identification of the specificity of phosphorylation states of the ctd recognized by ff domains has been challenging due to the weak binding . in vitro binding assays and nmr titration experiments
did not detect interaction with a ctd peptide with the ff domain arrays derived from prp40 ( 81 ) or ca150 ( 82 ) .
a binding constant of 50 m was reported for the mammalian homologue of prp40 , fbp11 , using isothermal titration calorimetry ( 83 ) but detailed information about the interaction between molecules is yet to be elucidated .
recognition of the phosphorylation states of the ctd is essential for mediation of the expression of genetic information .
communication of the ctd and histone codes provides an exquisite regulatory network for the precise control of transcription at multiple levels . some ctd interacting proteins function as global transcriptional regulators non - discriminatingly .
the inactivation of such molecules will lead to the shutdown of transcription machinery and eventually , cell death .
for example , the deletion of the ctd ser2 phosphatase fcp1 is lethal for yeast due to the abolishment of rna polymerase ii recycling ( 58 , 84 ) .
similar effects on yeast are observed when the ctd ser5 phosphatase ssu72 is eliminated ( 85 ) . on the other hand
, evidence has indicated that ctd - interacting proteins can control gene expression at an epigenetic level and regulate expression of specific genes based on timing and the developmental needs .
human scps are only found in higher eukaryotes and their expression is limited to neuronal stem cells or non - neural tissues .
consistent with their unique expression profile , scps are identified as a component of the neuronal chromatin remodeling complex , rest ( 61 ) .
the inactivation of scp activity leads to the inappropriate differentiation of neuronal stem cells ( 61 ) .
another example of epigenetic regulation of a ctd - interacting protein is the cdk8/cyclin c pair , which has been linked to transcriptional repression ( 48 ) .
histone methyltransferases set1 ( 75 ) and set2 ( 74 ) can identify the different phosphorylation stages of ctd .
the phosphorylation of ser7 of ctd itself also occurs in a gene - specific fashion ( 12 ) .
the apparent simple primary sequence of ctd integrates genetic and epigenetic information and plays a pivotal role in transcriptional activation or repression .
understanding how such coding is deciphered at the molecular level is essential to the interpretation of the central role of rna polymerase ii and its regulatory domains .
the application of ctd code provides a unique opportunity to engineer the transcriptional process in an epigenetic level in tissue - specificity manner .
there is no conflict of interest in the present study for any of the authors .
 in eukaryotic cells , the transcription of genes is accurately orchestrated both spatially and temporally by the c - terminal domain of rna polymerase ii ( ctd ) . 
 the ctd provides a dynamic platform to recruit different regulators of the transcription apparatus . 
 different posttranslational modifications are precisely applied to specific sites of the ctd to coordinate transcription process . 
 regulators of the rna polymerase ii must identify specific sites in the ctd for cellular survival , metabolism , and development . even though the ctd is disordered in the eukaryotic rna polymerase ii crystal structures due to its intrinsic flexibility , recent advances in the complex structural analysis of the ctd with its binding partners provide essential clues for understanding how selectivity is achieved for individual site recognition . 
 the recent discoveries of the interactions between the ctd and histone modification enzymes disclose an important role of the ctd in epigenetic control of the eukaryotic gene expression . 
 the intersection of the ctd code with the histone code discloses an intriguing yet complicated network for eukaryotic transcriptional regulation . 
Pin1 and the C-terminal domain (CTD)
CTD-interacting domain (CID) domain and the C-terminal domain (CTD)
Small C-terminal domain phosphatases (Scp)/Fcp phosphatases and the C-terminal domain (CTD)
mRNA capping enzyme Cgt1 and the C-terminal domain (CTD)
Nuclear magnetic resonance (NMR) structure of Set2
Concluding remarks
Conflict of interest and funding