pseudouridine (  ) is the most abundant post - transcriptional rna base modification and the enzymes responsible for this isomerization of uridine and the positions modified in the rnas , are conserved ( 14 ) .
this modification is important in the structure and function of small rnas ( 511 ) , including small nuclear rnas ( snrnas ) , cofactors in pre - mrna splicing ( 1216 ) .
one pathway is involved in the formation of  in rrna and snrna and requires dyskerin or its homologs ( cbf5p in yeast for example ) and rnp cofactors [ most often h / aca small nucleolar ribonucleoprotein particles ( snornps ) ] that enable one enzyme to recognize many different sites for modification on different substrates ( 1725 ) .
the other pathway for  formation employs site - specific  synthases that require no cofactors to recognize and modify the rna substrate .
a number of enzymes have been identified in this pathway and are grouped in six families that all share a common basic structure ( 4 ) .
guided pathway has received a great deal of attention because of its similarity to aspects of rna editing , but the site - specific pseudouridine synthases accomplish the same task , on more substrates , with fewer tools , arguably making them more interesting from an enzymatic perspective .
one of the most studied site - specific enzymes is pseudouridine synthase 1 ( pus1p ) ( 2635 ) .
pus1p modifies uridines in several regions of many different trnas ( 27,33,34 ) and one position in yeast u2 snrna ( 36 ) .
it is a transcriptional coactivator in nuclear receptor signaling , modifying steroid receptor activator rna ( sra ) ( 37,38 ) , and when mutated or missing , leads to the disorder mitochondrial myopathy and sideroblastic anemia with lactic acidemia ( mlasa ) ( 3942 ) . in vivo and in vitro pus1p modifies positions 1 , 26 , 27 and 28 in many trnas and positions 34 and 36 in the anticodons of certain intron - containing trnas ( 2628,33 ) .
in addition , yeast pus1p has been shown to modify positions 65 and 67 in vivo ( 34 ) and mouse ( mpus1p ) and human pus1p ( hpus1p ) modify position 30 in trnas , whereas yeast pus1p does not ( 29,33 ) .
the presence of the intron in yeast pre - trna(uau ) is required for the formation of  at positions 34 and 36 in the anticodon by pus1p in vitro ( 26,34 ) .
the intron allows for the formation of a stem that contains the uridines to be modified ( 26,34,43 ) .
residues that are modified in trna by pus1p are part of a base - paired stem , suggesting rna secondary structure is important for recognition by pus1p ( 26,27,3335 ) . even with all of the studies on pus1p , the only sequence consensus ( not requirement ) that has been proposed for pus1p is the presence of a pyrimidine nucleotide 3 of the site of modification ( 44 ) .
interestingly , yeast pus1p has been shown to modify position 44 on yeast u2 snrna , a position that is considered to be in a non - base paired region of u2 snrna ( 36 ) .
when mouse pus1p is expressed in a pus1 deleted yeast strain , the mouse enzyme can also modify yeast u2 snrna at the same position ( 33 ) .
however , the modification of u2 snrna in caenorhabditis elegans does not require pus1p , since the loss of the pus-1 gene product in worms results in the absence of  at known pus1p modification sites on trna , but has no effect on the modification of u2 snrna or other snrnas ( 28,45 ) .
however , the structure of a member of the same synthase family , bacterial trua , in the presence of trna substrates , has been revealed ( 46,47 ) .
trua modifies uridines at positions 3840 in many trnas and the equivalent activity in eukaryotes is associated with pseudouridine synthase 3 , another member of the trua family [ pus3p ; ( 48,49 ) ] .
the substrate trna binds across a dimer of trua ( 46,47 ) . the anticodon stem - loop ( asl ) of the trna , as well as the dihydrouridine ( d ) stem and the  elbow where the d and tc loops interact , contact the synthase ( 47 ) .
trua did not interact with the variable ( v ) stem - loop or the acceptor stem of the trna substrate ( 47 ) . in order to begin to understand how pus1p can modify several positions in trnas and
also modify other rnas such as u2 snrna and sra , we undertook an investigation of the sequence and structural requirements for hpus1p activity on human trna .
the activity of hpus1p with wild - type and point and deletion mutants of the trna substrate indicates there are structural requirements in the formation of  at position 28 in trna .
a minimum level of base pairing in the asl ( the location of the modification ) and the presence of an additional 3 stem - loop , constitute the minimal substrate for hpus1p .
even when all nucleotides in the asl stem other than u28 were changed in a single minimal substrate , but base pairing was retained , a near wild - type level of modification was observed .
site - directed mutagenesis of human trna ( uga ) was performed with a quikchange ii kit ( stratagene ) , utilizing primers containing the mutations and the phts plasmid [ human trna(uga ) ; gift of h. gross ( 50 ) ] to create the mutants .
the resulting constructs have a t7 rna polymerase promoter at the 5-end of the trna gene and a bstn1 site at the 3-end of the gene .
plasmid dna digested with bstn1 will yield trnas with cca at the 3-terminus after transcription ( 29,50 ) .
the primers used for mutagenesis are given below with just the forward primers listed ; the reverse primers are the inverse complement .
the forward primer for : 
 27c was 5cgagtggttaaggcgctggacttgaaatcca,27 g was 5cgagtggttaaggcggtggacttgaaatcca,28c was 5gagtggttaaggcgacggacttgaaatcc used with the 42 g mutant to create 28c/42g,29c was 5gagtggttaaggcgatcgacttgaaatccatt,30c was 5gtggttaaggcgatgcacttgaaatccattg,31c was 5gtggttaaggcgatggccttgaaatccattgg,39 g was 5cgatggacttgaaagccattggggtc,40 g was 5gatggacttgaaatgcattggggtctc,41 g was 5gacttgaaatcgattggggtctcc , and used in the 29c/41 g mutant starting with 29c template,42c was 5ggacttgaaatcccttggggtctcc,42 g was 5ggacttgaaatccgttggggtctcc,43 g was 5ggacttgaaatccagtggggtctcc , and used in the 27c/43 g mutant starting with 27c template and in 27g/43 g mutant starting with the 27 g template,30c/40 g was 5gatgcacttgaaatgcattggggtctc , starting with the 30 g template,31c/39 g was 5cgatggccttgaaagccattggggtc , starting with the 31c template, d stem - loop was 5gtagtcgtggcgatggacttgaaatcc, d stem - loop with 28c/42 g was 5gtagtcgtggcgacggacttgaaatccgttggggtctcccc .
27c was 5cgagtggttaaggcgctggacttgaaatcca , 27 g was 5cgagtggttaaggcggtggacttgaaatcca , 28c was 5gagtggttaaggcgacggacttgaaatcc used with the 42 g mutant to create 28c/42 g , 29c was 5gagtggttaaggcgatcgacttgaaatccatt , 30c was 5gtggttaaggcgatgcacttgaaatccattg , 31c was 5gtggttaaggcgatggccttgaaatccattgg , 39 g was 5cgatggacttgaaagccattggggtc , 40 g was 5gatggacttgaaatgcattggggtctc , 41 g was 5gacttgaaatcgattggggtctcc , and used in the 29c/41 g mutant starting with 29c template , 42c was 5ggacttgaaatcccttggggtctcc , 42 g was 5ggacttgaaatccgttggggtctcc , 43 g was 5ggacttgaaatccagtggggtctcc , and used in the 27c/43 g mutant starting with 27c template and in 27g/43 g mutant starting with the 27 g template , 30c/40 g was 5gatgcacttgaaatgcattggggtctc , starting with the 30 g template , 31c/39 g was 5cgatggccttgaaagccattggggtc , starting with the 31c template ,  d stem - loop was 5gtagtcgtggcgatggacttgaaatcc ,  d stem - loop with 28c/42 g was 5gtagtcgtggcgacggacttgaaatccgttggggtctcccc .
the digested plasmid dnas were transcribed with t7 rna polymerase in the presence of 25  ci [ 5-h]-utp ( 11.1 or 23.9  ci / mmol , moravek ) or 25  ci [ 5,6-h]-utp ( 38  ci / mmol , moravek ) with 100  m cold utp and 1.0  mm , non - labeled , atp , gtp and ctp ( 51 ) .
additional rna substrates were synthesized using a sense t7 dna oligonucleotide , an antisense template oligonucleotide and high concentration t7 rna polymerase , as described ( 52,53 ) .
the oligodeoxynucleotides used for this type of synthesis are listed below , note that the oligodeoxynucleotides that serve as the template for synthesis are antisense and have antisense t7 primer sequence at the 3-end .
the template for the : 
 asl stem - loop was 5aatggatttcaagtccatctatagtgagtcgtatta , asl & d stem - loops was 5atggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl & v stem - loops was 5ggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , d , & v stem - loops was 5ggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl , d , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta, tc stem - loop was 5tggcgtagtcggcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta, v stem - loop was 5 tggcgtagtcggcaggattcgaacctgcgaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta.asl & tc stem - loop mini - substrate required the following oligodeoxynucleotide template : 5gcaggattcgaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta .
the small size of this mini - substrate and the fact that the first nucleotide transcribed is a 
the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta,28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta,27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta,27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta,27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta,27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta,27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta,55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta,5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta .
asl stem - loop was 5aatggatttcaagtccatctatagtgagtcgtatta , asl & d stem - loops was 5atggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl & v stem - loops was 5ggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , d , & v stem - loops was 5ggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl , d , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta ,  tc stem - loop was 5tggcgtagtcggcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta ,  v stem - loop was 5 tggcgtagtcggcaggattcgaacctgcgaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta .
asl & tc stem - loop mini - substrate required the following oligodeoxynucleotide template : 5gcaggattcgaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta .
the small size of this mini - substrate and the fact that the first nucleotide transcribed is a  g , allows for efficient synthesis of rnas in vitro .
the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta , 28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta , 27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta , 27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta , 27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta , 27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta , 27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta , 55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , 5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . before incubation with hpus1p ,
the rna substrates were heated to 78c for 3  min and allowed to cool slowly to at least 37c ( 3040  min ) .
substrate reactions with the  synthases were carried out in 50  mm ammonium chloride , 5  mm dtt , 25  mm tris ( ph 7.5 ) and 1  mm mgcl2 . the amount of  synthase used was ~200  ng at 37c for the times indicated .
norit a charcoal binds all of the h counts in the sample except those that have been 
released to water in the process of forming . the released counts are separated from the charcoal - bound counts using a spin - x column ( costar ) , the filtrate is mixed with scintillation fluid , and the released h counts are measured on a scintillation counter .
the equilibrium binding assays were carried out as described ( 32 ) using hpus1p concentrations of 0 , 50 , 100 , 200 , 400 and 800  nm in 50  l reactions , with the h - labeled rna substrates at 500  pm . the rnas were synthesized as described above except that the cold utp concentration in the synthesis reaction was 250  m . a slot - blot apparatus ( biorad ) was used for filtration of the samples , the resulting filters were air dried , cut into appropriate pieces and counted in scintillation fluid .
site - directed mutagenesis of human trna ( uga ) was performed with a quikchange ii kit ( stratagene ) , utilizing primers containing the mutations and the phts plasmid [ human trna(uga ) ; gift of h. gross ( 50 ) ] to create the mutants .
the resulting constructs have a t7 rna polymerase promoter at the 5-end of the trna gene and a bstn1 site at the 3-end of the gene .
plasmid dna digested with bstn1 will yield trnas with cca at the 3-terminus after transcription ( 29,50 ) .
the primers used for mutagenesis are given below with just the forward primers listed ; the reverse primers are the inverse complement .
the forward primer for : 
 27c was 5cgagtggttaaggcgctggacttgaaatcca,27 g was 5cgagtggttaaggcggtggacttgaaatcca,28c was 5gagtggttaaggcgacggacttgaaatcc used with the 42 g mutant to create 28c/42g,29c was 5gagtggttaaggcgatcgacttgaaatccatt,30c was 5gtggttaaggcgatgcacttgaaatccattg,31c was 5gtggttaaggcgatggccttgaaatccattgg,39 g was 5cgatggacttgaaagccattggggtc,40 g was 5gatggacttgaaatgcattggggtctc,41 g was 5gacttgaaatcgattggggtctcc , and used in the 29c/41 g mutant starting with 29c template,42c was 5ggacttgaaatcccttggggtctcc,42 g was 5ggacttgaaatccgttggggtctcc,43 g was 5ggacttgaaatccagtggggtctcc , and used in the 27c/43 g mutant starting with 27c template and in 27g/43 g mutant starting with the 27 g template,30c/40 g was 5gatgcacttgaaatgcattggggtctc , starting with the 30 g template,31c/39 g was 5cgatggccttgaaagccattggggtc , starting with the 31c template, d stem - loop was 5gtagtcgtggcgatggacttgaaatcc, d stem - loop with 28c/42 g was 5gtagtcgtggcgacggacttgaaatccgttggggtctcccc .
27c was 5cgagtggttaaggcgctggacttgaaatcca , 27 g was 5cgagtggttaaggcggtggacttgaaatcca , 28c was 5gagtggttaaggcgacggacttgaaatcc used with the 42 g mutant to create 28c/42 g , 29c was 5gagtggttaaggcgatcgacttgaaatccatt , 30c was 5gtggttaaggcgatgcacttgaaatccattg , 31c was 5gtggttaaggcgatggccttgaaatccattgg , 39 g was 5cgatggacttgaaagccattggggtc , 40 g was 5gatggacttgaaatgcattggggtctc , 41 g was 5gacttgaaatcgattggggtctcc , and used in the 29c/41 g mutant starting with 29c template , 42c was 5ggacttgaaatcccttggggtctcc , 42 g was 5ggacttgaaatccgttggggtctcc , 43 g was 5ggacttgaaatccagtggggtctcc , and used in the 27c/43 g mutant starting with 27c template and in 27g/43 g mutant starting with the 27 g template , 30c/40 g was 5gatgcacttgaaatgcattggggtctc , starting with the 30 g template , 31c/39 g was 5cgatggccttgaaagccattggggtc , starting with the 31c template ,  d stem - loop was 5gtagtcgtggcgatggacttgaaatcc ,  d stem - loop with 28c/42 g was 5gtagtcgtggcgacggacttgaaatccgttggggtctcccc .
the digested plasmid dnas were transcribed with t7 rna polymerase in the presence of 25  ci [ 5-h]-utp ( 11.1 or 23.9  ci / mmol , moravek ) or 25  ci [ 5,6-h]-utp ( 38  ci / mmol , moravek ) with 100  m cold utp and 1.0  mm , non - labeled , atp , gtp and ctp ( 51 ) .
additional rna substrates were synthesized using a sense t7 dna oligonucleotide , an antisense template oligonucleotide and high concentration t7 rna polymerase , as described ( 52,53 ) .
the oligodeoxynucleotides used for this type of synthesis are listed below , note that the oligodeoxynucleotides that serve as the template for synthesis are antisense and have antisense t7 primer sequence at the 3-end .
the template for the : 
 asl stem - loop was 5aatggatttcaagtccatctatagtgagtcgtatta , asl & d stem - loops was 5atggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl & v stem - loops was 5ggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , d , & v stem - loops was 5ggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl , d , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta, tc stem - loop was 5tggcgtagtcggcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta, v stem - loop was 5 tggcgtagtcggcaggattcgaacctgcgaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta.asl & tc stem - loop mini - substrate required the following oligodeoxynucleotide template : 5gcaggattcgaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta .
the small size of this mini - substrate and the fact that the first nucleotide transcribed is a 
the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta,28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta,27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta,27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta,27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta,27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta,27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta,27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta,55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta,5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta .
asl stem - loop was 5aatggatttcaagtccatctatagtgagtcgtatta , asl & d stem - loops was 5atggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl & v stem - loops was 5ggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatctatagtgagtcgtatta , asl , d , & v stem - loops was 5ggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta , asl , d , v , & tc stem - loops was 5gcaggattcgaacctgcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggctatagtgagtcgtatta ,  tc stem - loop was 5tggcgtagtcggcgcggggagaccccaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta ,  v stem - loop was 5 tggcgtagtcggcaggattcgaacctgcgaatggatttcaagtccatcgccttaaccactcggccacgactactatagtgagtcgtatta .
asl & tc stem - loop mini - substrate required the following oligodeoxynucleotide template : 5gcaggattcgaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta . the small size of this mini - substrate and the fact that the first nucleotide transcribed is a  g , allows for efficient synthesis of rnas in vitro .
the following mutations were incorporated into the oligodeoxynucleotide template to generate variations of the asl & tc stem - loop mini - substrate : ggacg starting at 61 was 5cgtccattcgaacctgcgccaatggatttcaagtccatcatagtgagtcgtatta , aagcuua in tc loop was 5gcaggtaagcttcctgcgccaatggatttcaagtccatcatagtgagtcgtatta , 28c/42 g was 5gcaggattcgaacctgcgccaacggatttcaagtccgtctatagtgagtcgtatta , 27c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtccagctatagtgagtcgtatta , 27c/42c was 5gcaggattcgaacctgcgccaagggatttcaagtccagctatagtgagtcgtatta , 27c/29c/39 g was 5gcaggattcgaacctgcgccaatggctttcaagtcgagctatagtgagtcgtatta , 27c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtccagctatagtgagtcgtatta , 27c/29c/42 g was 5gcaggattcgaacctgcgccaagggatttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40 g was 5gcaggattcgaacctgcgccaatgcctttcaagtcgagctatagtgagtcgtatta , 27c/29c/39g/40g/42c was 5gcaggattcgaacctgcgccaaggcctttcaagtcgagctatagtgagtcgtatta , 55 & 56 was 5gcaggattcacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , 5558 was 5gcaggatacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , gcaa inserted after 55 was 5gcaggattcgttgcaacctgcgccaatggatttcaagtccatctatagtgagtcgtatta , y for y r for r stem mini - substrate mutant was 5gcaggattcgaacctgcgccagcaagtttcaagcttacctatagtgagtcgtatta . before incubation with hpus1p , the rna substrates were heated to 78c for 3  min and allowed to cool slowly to at least 37c ( 3040  min ) .
substrate reactions with the  synthases were carried out in 50  mm ammonium chloride , 5  mm dtt , 25  mm tris ( ph 7.5 ) and 1  mm mgcl2 . the amount of  synthase used was ~200  ng at 37c for the times indicated .
norit a charcoal binds all of the h counts in the sample except those that have been 
released to water in the process of forming . the released counts are separated from the charcoal - bound counts using a spin - x column ( costar ) , the filtrate is mixed with scintillation fluid , and the released h counts are measured on a scintillation counter .
the equilibrium binding assays were carried out as described ( 32 ) using hpus1p concentrations of 0 , 50 , 100 , 200 , 400 and 800  nm in 50  l reactions , with the h - labeled rna substrates at 500  pm .
the rnas were synthesized as described above except that the cold utp concentration in the synthesis reaction was 250  m . a slot - blot apparatus ( biorad )
was used for filtration of the samples , the resulting filters were air dried , cut into appropriate pieces and counted in scintillation fluid .
as an initial investigation into the substrate requirements for hpus1p , it was important to choose a known pus1p substrate trna that has only one site modified by the enzyme ( 29,35 ) , to simplify the analysis of the activity .
a trna that is typical in structure and has a v stem - loop that was > 6  nt , was preferred in order to be able to test the contribution of all structural components to substrate recognition by hpus1p . in addition , a trna with traditional watson - crick base - pairing in the asl stem was desired .
the trna sequence database , trnadb ( 56,57 ) , was consulted to compile consensus sequences for the asl stem where a  or u is found at position 28 in sequenced trnas from the fungi and metazoan group in trnadb .
at least 36 trna sequences were compiled to generate each consensus shown in figure 1b .
first , with a  at position 28 , g30c40 is the most conserved base pair ( > 90% conserved ) but when a u is found at position 28 , this sequence is not as well conserved .
therefore , one might expect that disruption of this base pair or replacement with the inverse ( c30g40 ) will affect modification .
second , for sequenced trnas that have a  at 28 , the preferred 2743  bp is u27a43 , whereas with those trnas with a u at 28 , the preferred base pair is c27g43 .
third , a a31u39 base pair is more common in those asl stems that have a  at position 28 than those with a u at position 28 .
finally , the base pairs 2941 and 3139 , below the 2842  bp , are watson - crick only but the rest of the base pairs in the stem can have g - u pairing in either consensus . in the selection of a trna , a good match to this consensus was preferred , and human trna(uga ) meets most of the criteria .
the only difference is that this trna has an a27u43 base pair instead of a u  a base pair at that position ( figure 1a ) . 

( a ) the sequence and proposed secondary structure of human trna(uga ) ( 50 ) .
the major aspects of the secondary structure are labeled on the diagram and it is numbered without including most of the variable stem - loop . the single site of pus1p modification , position 28 , is boxed .
( b ) consensus sequences for sequenced trnas from the fungi and metazoa group in the trnadb ( 57 ) that have a  or a u at position 28 .
a solid line between nucleotides indicates watson - crick pairs only and a dash line indicates g  u pairs ( < 50% ) are also found at this position .
( a ) the sequence and proposed secondary structure of human trna(uga ) ( 50 ) .
the major aspects of the secondary structure are labeled on the diagram and it is numbered without including most of the variable stem - loop . the single site of pus1p modification , position 28 , is boxed .
( b ) consensus sequences for sequenced trnas from the fungi and metazoa group in the trnadb ( 57 ) that have a  or a u at position 28 . the figure is presented in the output style from trnadb .
a solid line between nucleotides indicates watson - crick pairs only and a dash line indicates g  u pairs ( < 50% ) are also found at this position .
the point mutations in the asl of full - length trna are diagrammed in figure 2h and were obtained by site - directed mutagenesis of the plasmid containing wild - type human trna [ phts , gift from h. gross ; ( 50 ) ] followed by in vitro transcription of linear templates ( see  materials and methods section ) .
time course analyses of the in vitro activity of the substrate rnas and hpus1p are presented in figure 2 ( panels a 
g ) , with the same wild - type trna curve in all panels ( figure 2a  g ) .
the formation of  in this trna by hpus1p is reduced to background levels ( 0.02  mol /mol rna ) if the u at position 28 is changed to c and a42 is changed to g to preserve base pairing ( table 2 , second experiment ) .
this shows that the only u converted to  by hpus1p is the one at position 28 .
the panels in figure 2 each contain the wild - type trna curve and either two or three curves generated with trna mutant substrates .
this style allows for a simplification of the presentation and for the focus in most panels to be on 1  bp at a time . 

figure 2.time course experiments with single and double mutants in the asl of human trna .
( a  g ) , h - labeled rnas were incubated with hpus1p for the times indicated and the amount of  formed assayed .
each panel has the same time course of wild - type ( wt ) trna values .
each mutant is plotted separately and standard deviation ( sd ) bars are displayed for all time points .
( h ) diagram of asl point mutations made in the context of full - length trna that are presented in panels a 
( a  g ) , h - labeled rnas were incubated with hpus1p for the times indicated and the amount of  formed assayed .
each panel has the same time course of wild - type ( wt ) trna values .
each mutant is plotted separately and standard deviation ( sd ) bars are displayed for all time points .
( h ) diagram of asl point mutations made in the context of full - length trna that are presented in panels a 
when the a at 27 is changed to c or g ( figure 2a ) , the levels of  formed are not significantly different from wild - type trna .
the curve that results when the u at 43 is changed to g is distinct from the wild - type trna curve but the difference is not statistically significant because of the relatively large error bars ( sd ) .
mutation of the a at the position across from u28 to either c or g has little effect on time course of  formed ( figure 2b ) .
even though the a at position 42 is preferred ( figure 1b ) , the formation of 28 does not require canonical or even u 
g base pairing , something unanticipated given the consensus sequence ( figure 1b ) . when g29 is changed to c or c41
is changed to g ( figure 2c ) , there is a significant decrease in  formation for both mutations in the formation of  at 28 .
however , when the base pair is reformed , but is now c - g in the case of the double mutant 29c/41 g , the curve obtained is indistinguishable from that of wild - type trna ( figure 2f ) .
this introduction of a base - paired pyrimidine 3 to u28 did not enhance the formation of  in the substrate , an expectation if a pyrimidine is preferred at position 29 , as suggested in the introduction .
the consensus sequence shown in figure 1b revealed the g30c40 base pair to be highly conserved in the putative pus1p substrates with a  at position 28 .
it was expected that disruption of this base pair would affect the formation of  by hpus1p .
in fact , the 30c and 40 g mutants have significantly lower amounts of  formed than wild - type trna ( figure 2d ) .
if the base pair is reformed but inverted , as with the 30c/40 g double mutant , the time course of  formed is the same as that of wild - type trna ( figure 2f ) .
this result argues against the requirement for a g at position 30 , even though the 28 consensus suggests it ( figure 1b ) .
when the base pair at a31u39 is disrupted by either the 31c or 39 g point mutations , the results are interesting .
the curve of  formation seen with the 31c substrate is significantly different from wild - type trna ( figure 2e ) , but with the 39 g mutant the curve mimics that seen with wild - type trna ( figure 2e ) .
reforming the base pair with the 31c/39 g double mutant results in a curve that matches that of wild - type trna ( figure 2f ) .
the only difference between the 28 consensus stem ( figure 1b ) and the stem of the trna ( figure 1a ) is the 2743  bp , which is a  u in trna .
the  formation curves for the 27c , 27 g and 43 g mutants were not significantly different from wild - type trna ( figure 2a ) .
this would suggest that the enzyme does not require a base pair at 2743 , and may in fact be indifferent to the presence of a base pair .
that statement was supported by the  formation curve seen with the 27g/43 g double mutant , which was not significantly different from the curve seen with wild - type trna ( figure 2 g ) , even though there is no canonical base pair formed .
the results with the double mutant 27c/43 g , which would form a stronger base pair than the wild - type a  u , are also not significantly different from wild - type trna ( figure 2 g ) even though this mutant now mimics the u28 consensus in figure 1b .
equilibrium dissociation constants ( kd ) were determined for wild - type trna and the single- and double - mutant substrates found in the panels of figure 2 .
the means and standard deviations of two separate kd determinations are presented in table 1 and that of wild - type trna ( 240  nm ) agrees with a previous determination of the kd ( 250  nm ) of hpus1p and yeast pre - trna ( 29 ) .
all of the mutants have a higher mean kd than wild - type , with all but 41 g and 43 g > 300  nm .
there is not a correlation between the  formation levels and the kds for the mutants .
it is interesting to note that a trna that is not a substrate for hpus1p , such as the 28c/42 g double mutant , binds to the enzyme with a mean kd of 332  nm .
this was also as seen previously with yeast pus1p and a non - substrate trna ( 31 ) . 

table 1.equilibrium dissociation constants ( kd ) of hpus1p with various rnasrna substratekd mean ( sd)wild - type trna240 ( 14 )  nmsingle point mutants in trna  27c328 ( 46 )  nm  27g370 ( 14 )  nm  29c408 ( 11 )  nm  30c335 ( 120 )  nm  31c358 ( 53 )  nm  39g380 ( 28 )  nm  40g305 ( 49 )  nm  41g282 ( 18 )  nm  42c360 ( 57 )  nm  42g338 ( 46 )  nm  43g248 ( 32 )  nmdouble mutants in trna  27c/43g302 ( 39 )  nm  27g/43g375 ( 7 )  nm  28c/42g332 ( 18 )  nm  29c/41g328 ( 74 )  nm  30g/40c345 ( 21 )  nm  31c/39g315 ( 92 )  nmlisted are the means and standard deviations ( sd ) of two separate binding experiments of hpus1p and the substrates listed ( see  materials and methods section for details ) .
equilibrium dissociation constants ( kd ) of hpus1p with various rnas listed are the means and standard deviations ( sd ) of two separate binding experiments of hpus1p and the substrates listed ( see  materials and methods section for details ) . in order to identify additional recognition elements of the hpus1p substrate , stem or stem - loop deletions of , and mini - substrates derived from trna , were tested in the  formation assay .
these mutant substrates were generated either by site - directed mutagenesis of phts and subsequent in vitro transcription or by in vitro transcription using oligodeoxynucleotide templates [ see  materials and methods section ; ( 53 ) ] .
it is understood that , with the deletion of whole stem - loops or the creation of mini - substrates , the tertiary structure of the entire trna is affected , but surprisingly , many of these mutants can function as substrates for hpus1p .
the results for 120  min incubations are listed in table 2 relative to the activity seen with wild - type trna .
if the d stem - loop is missing in the substrate , there is no decrease in hpus1p activity at position 28 .
if the u at position 28 in the d stem - loop mutant is changed to c ( and the a at 42 is changed to g ) so that  can not be formed at position 28 , there is essentially no  formation ( 2% of wild - type trna  formation , see table 2 ) .
this result indicates the level of  formation seen with the d stem - loop mutant is not due to  formation at an additional site created by an altered secondary structure in the deletion mutant .
if the tc stem - loop is missing from the trna , there is a significant reduction in the formation of  at u28 ( 15% of wild - type trna levels , table 2 ) but the reduction is not as substantial when the v stem - loop is missing ( 67% of wild - type , table 2 ) . 

table 2.activity of hpus1p on trna(uga ) point and deletion mutants and mini - substratesrna substratemole /mole rna ( sd)percent of wild - type ( sd)wild - type human trna0.60 ( 0.08)100 ( 13) tc stem - loop ( deleted nucleotides 5973)0.09 ( 0.05)15 ( 8) variable stem - loop ( deleted nucleotides 4556)0.40 ( 0.08)67 ( 13)wild - type human trna0.43 ( 0.07)100 ( 16)28c/42 g ( no  formation expected)0.02 ( 0)5 ( 0) d stem - loop ( deleted nucleotides 1124)0.44 ( 0.16)102 ( 37) d stem - loop with 28c/42g0.01 ( 0.01)2 ( 2)wild - type human trna0.68 ( 0.02)100 ( 3)asl stem loop alone ( includes nucleotides
2643)0.02 ( 0.01)3 ( 1)asl & d stem - loops ( includes nucleotides 1043)0.07 ( 0.01)10 ( 1)asl & v stem - loops ( includes nucleotides 2647)0.05 ( < 0.01)7 ( 1)asl , d , & v stem - loops ( includes nucleotides 1047)0.14 ( < 0.01)21 ( 1)asl , v , & tc stem - loops ( includes nucleotides
2665)0.48 ( 0.04)71 ( 6)asl , d , v , & tc stem - loops ( includes nucleotides 765)0.64 ( 0.16)94 ( 24)activities are from 2-h incubations , an average of two assays , with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type trna(uga ) with the standard deviation ( sd ) also reported as percent of wild - type .
these results are grouped as separate experiments with a separate wild - type control for each experiment .
activity of hpus1p on trna(uga ) point and deletion mutants and mini - substrates activities are from 2-h incubations , an average of two assays , with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type trna(uga ) with the standard deviation ( sd ) also reported as percent of wild - type .
these results are grouped as separate experiments with a separate wild - type control for each experiment .
when a mini - substrate of just the asl stem - loop was incubated with hpus1p ,  formation was at background levels for 2  h of incubation ( 3% of wild - type levels , table 2 ) .
in addition , if either the d stem - loop or the v stem - loop was combined with the asl stem - loop , there was little increase in the amount of  formed ( 10% and 7% , respectively , of the  formation seen in 120  min with wild - type trna , table 2 ) .
this implies that adding a single stem - loop to the 5 or 3 side of the asl stem - loop does not constitute an efficient substrate for hpus1p .
if both the d and v stem - loops are added to the asl stem - loop there is a noticeable increase in  formed but it is still low ( 21% ) compared to wild - type trna .
when the tc stem - loop was combined with the v and asl stem - loops , there was a substantial level of hpus1p activity ( 71% of wild - type trna levels of  ) . with just the acceptor stem missing from trna ( asl , d , v and tc stem - loops present ) ,
the activity is essentially the same as with wild - type trna ( 94% of wild - type  formation at 120  min , table 2 ) .
these results suggest that the secondary structure of the trna is very important for the modification at position 28 , and a minimal substrate could be created by simply connecting the asl and tc stem - loop as diagrammed ( figure 3a ) .
the numbering on the diagram is consistent with full - length trna to allow comparisons with the results reported in figure 2 and table 2 .
the level of  formation at position 28 in this minimal substrate ( wild - type asl & tc mini - substrate ) was ~0.60  mol /mol rna after 2  h of incubation , a substantial level of modification ( table 3 ) .
a mini - substrate incorporating a 28c/42 g double mutation was used in a separate experiment with little or no  formation observed ( table 3 ) , providing evidence that in this mini - substrate , the u at position 28 is the only one being converted to  , as expected . 

( a ) predicted structure of the asl & tc stem loop mini - substrate , retaining the numbering found on the full - length trna and indicating the position of the  formed .
the structure for the y for y r for r stem mini - substrate mutant and the c28 mutant are also shown .
( b ) diagram of the possible base pairing in the asl stem mutants in the mini - substrate listed in table 3 .
the activities in percent of the level observed with wild - type asl & tc stem - loop mini - substrate without the sd are listed above the diagrams .
table 3.activity of hpus1p on the wild - type asl & tc stem - loop mini - substrate and mutantsrna substratemole /mole rna ( sd)mole /mole rna ( sd)percent of wild - type ( sd)wild - type asl & tc stem - loop mini - substrate0.60 ( 0.11)0.54 ( 0.16)100 ( 18 ) ( 30)28c/42 g ( no u at expected site of  formation)0.01 ( 0.01)2 ( 2)27c/42c0.48 ( 0.04)80 ( 7)27c/39g0.24 ( 0.05)40 ( 8)27c/29c/39g0.10 ( 0.03)17 ( 5)27c/29c/42c0.04 ( 0.01)7 ( 2)27c/39g/40g<0.01 ( 0.01)2 ( 2)27c/29c/39g/40g0.02 ( 0.03)3 ( 5)27c/29c/39g/40g/42c0.02 ( < 0.01)3 ( 2)55 & 560.37 ( 0.10)62 ( 17)55580.17 ( 0.06)28 ( 10)gcaa inserted between 55 and 560.46 ( 0.04)77 ( 7)wild - type asl & tc stem - loop mini - substrate0.30 ( 0.04)100 ( 13)ggacg starting at 610.22 ( 0.03)73 ( 10)aagcuua in tc loop0.34 ( 0.04)113 ( 13)activities are from 2-h incubations , an average of two assays with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type .
all of the listed mutants are in the context of the asl & tc stem - loop mini - substrate .
( a ) predicted structure of the asl & tc stem loop mini - substrate , retaining the numbering found on the full - length trna and indicating the position of the  formed .
the structure for the y for y r for r stem mini - substrate mutant and the c28 mutant are also shown .
( b ) diagram of the possible base pairing in the asl stem mutants in the mini - substrate listed in table 3 .
the activities in percent of the level observed with wild - type asl & tc stem - loop mini - substrate without the sd are listed above the diagrams .
activity of hpus1p on the wild - type asl & tc stem - loop mini - substrate and mutants activities are from 2-h incubations , an average of two assays with duplicate samples , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type .
all of the listed mutants are in the context of the asl & tc stem - loop mini - substrate .
concentrating on the asl stem , a number of mutant substrates were synthesized that pick up where the previous mutations in the full length trna ( figure 2 and table 2 ) left off .
the asl stem of trna has five watson - crick base pairs and the mini - substrate mutants listed in table 3 were designed to eliminate two or more of those base pairs in each mutant .
the base pairs that may still form in each mutant are diagrammed in figure 3b and the levels of activity , as a percentage of the activity observed with the wild - type asl & tc mini - substrate , are given in table 3 and figure 3b . when there is no base - pairing in the stem there is little formation of  , and the possibility of 1 or even 2  bp in the stem , does not substantially elevate the amount of  formed
. however , with the 27  c/42c mutant substrate , which has 3  bp in the stem below the site of modification , 80% of the  level seen with the wild - type asl & tc mini - substrate was observed .
the level of  formation by hpus1p on the 27c/39 g mutant substrate was only 40% , even though it too has 3  bp in the stem .
with respect to the tc stem - loop , if the nucleotides at 55 and 56 are deleted in this asl & tc mini - substrate , there is a decrease in  formation relative to the wild - type asl & tc mini - substrate ( 62% of wild - type trna , table 3 ) .
if the size of the tc loop is reduced further ( 5558 ) the resulting substrate is a poor one , just 28% of wild - type levels of  were observed ( table 3 ) .
if 4  nt ( gcaa ) are inserted into the tc loop to make it larger , the substrate still allows for a significant amount of  to be formed ( 77% of wild - type asl & tc mini - substrate levels ) . in a separate experiment ,
if watson - crick base - pairing is disrupted in the tc stem , with the sequence ggacg starting at nucleotide 61 , the level of hpus1p activity was also substantial ( 73% or wild - type asl & tc mini - substrate levels ) , suggesting a stem loop is not required . if the sequence of the tc loop was replaced with its complement ( aagcuua ) , the amount of  formed at position 28 was 113% of wild - type asl & tc mini - substrate levels , suggesting that any loop of at least 5  nt will suffice . to summarize the results from the mutation and deletion studies , the levels of  formation with
the single and double mutants in the context of the full - length trna suggest that the sequence of the asl stem is not an absolute determinant of hpus1p activity , except of course that there must be a u at position 28 to modify ( tables 2 and 3 ) .
the preference for a pyrimidine 3 to the site of modification ( at position 29 ; see 
introduction section ) was not confirmed since even though the 29  c single mutant showed a decreased amount of  formed and the double mutant 29  c/41  g exhibited an almost identical curve as wild - type trna , suggesting that a pyrimidine is not preferred , only the base pair .
the point mutations in full - length trna show that the base pairing below the modified position ( u28 ) affects  formation , whereas the 27  a 
the results with the asl & tc stem loop mini - substrate mutants also showed that the base pairs below u28 were the most critical .
the sequence of the stem nucleotides is of less importance than the presence of base pairs , which agrees with the fact that known substrates of pus1p exhibit no absolute sequence requirement ( 26,27,3335 ) . in terms of the requirements for a particular secondary structure of trna ,
the d - stem loop and acceptor stem are dispensable for  formation at u28 .
in fact , a single stem loop , 3 to the asl , is all that is required for substantial  formation at position 28 . the sequence , and possibly the structure of this additional rna , is not constrained .
given the above summary of the mutation experiments , a mini - substrate where all of the nucleotides in the asl stem have been changed , except of course for the u at position 28 which is modified , should be a substrate for hpus1p .
a substrate fitting this description ( figure 3a ) , along with another where the u at position 28 was changed to c , were incubated with hpus1p and assayed for activity .
the y for y r for r stem mini - substrate mutant has a replaced by g , g replaced by a , c replaced by u , and u replaced by c when compared with the wild - type asl & tc mini - substrate , except at position 28 .
the 28  c mutant has complete replacement of the stem sequence , and should not be modified by hpus1p .
the results with these substrates , as well as with the wild - type asl & tc mini - substrate , are presented in table 4 and show that this mutant is modified by hpus1p ( 0.33  mol /mol rna ) at 80% of wild - type levels for this experiment .
in addition , the 28c mutant is not modified ( 0.01  mol /mol rna ; 2% of wild - type levels ) , proving that only the u at position 28 is modified .
so , even though all the asl stem nucleotides in the mutant are different from the wild - type trna asl stem , and there is a u28g42 base pair in the mutant , the u at position 28 is converted to  at near wild - type levels . 

table 4.activity of hpus1p with wild - type asl & tc stem - loop mini - substrate and stem sequence mini - substrate mutantsrna substratemole /mole rna ( sd)percent of wild - type ( sd)wild - type asl & tc stem - loop mini - substrate0.41 ( 0.05)100 ( 12)y for y r for r stem mini - substrate mutant 27g/29a/30a/31g/39c/40u/41u/42g/43c0.33 ( 0.02)80 ( 5)y for y r for r stem mini - substrate 28c mutant 27g/28c/29a/30a/31g/39c/40u/41u/42g/43c0.01 ( 0.01)2 ( 2)activities are from 2-h incubations , an average of three separate assays , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type .
all of the listed mutants are in the context of the asl & tc stem - loop mini - substrate .
activity of hpus1p with wild - type asl & tc stem - loop mini - substrate and stem sequence mini - substrate mutants activities are from 2-h incubations , an average of three separate assays , and reported as mol /mol rna and as the percent of the level seen with wild - type asl & tc stem loop mini substrate , with the standard deviation ( sd ) also reported as percent of wild - type .
all of the listed mutants are in the context of the asl & tc stem - loop mini - substrate .
a number of studies have determined the sites in trnas that are modified by pus1p ( 26,27,29,33,34 ) .
yeast and mammalian pus1p are the most extensively studied and in vivo and in vitro , the enzymes are responsible for the formation of  at positions 27 and 28 in many trnas , at 34 and 36 in certain intron containing trnas , and at position 1 in a few substrates ( 29,33,34 ) .
the yeast enzyme can also modify uridine at positions 26 , 65 and 67 in vivo but not in vitro ( 34 ) . when the mouse enzyme was expressed in a pus1 yeast strain , position 26
was not modified , and the modification states of positions 65 and 67 were not determined ( 33 ) .
sequenced human trnas do not have  at positions 26 , 65 , or 67 ( 58 ) , so it is unlikely mpus1p or hpus1p exhibit activity at these sites in vivo .
the mammalian enzymes do modify position 30 in a few trnas but the yeast enzyme lacks this activity ( 29,33 ) .
for the purposes of the discussion of the current results , the modification at positions 27 and 28 will be considered , since these are formed in vitro in a number of substrates by the mammalian enzymes ( 27,33,35 ) . nearly all ( 17 out of 18 ) of the sequenced yeast and human trnas that have a u at position 27 have converted it to  ( 58 ) . however , in the six sequenced yeast trnas that have a u at position 28 , the three that have a 27c43 g base pair lack  at position 28 , whereas , the other three have a 2743a base pair and a  at position 28 ( 58 ) .
this completely agrees with the two consensus sequences shown in figure 1b . in the sequenced human trnas with a u at 28 ( eight total )
three of the trnas have a 2743a base pair , two have a  at position 28 , but one does not
. a human trna with an 27a43u base pair does have a  at position 28 ( 58 ) , suggesting that an u  a or a  u base pair at 2743 allows for  formation at 28 , whereas a 27c43 g base pair is not conducive to  formation at position 28 .
however , with the four remaining trnas , which have a 27c43 g base pair , two have a  at position 28 and two do not ( 58 ) .
the current finding that the loss of base pairing at , or the presence of , a c  g base pair at 2743 does not significantly affect the formation of  at position 28 in trna , correlates with the data from human trnas .
human mitochondrial trna is known to be a substrate of hpus1p in vivo ( 41 ) but the sequence of the stem only matches the 28 consensus at the 2743 and 2842  bp .
finally , the y for y r for r stem mini - substrate mutant is an efficient substrate for hpus1p in vitro , even though the sequence in the stem does not match the consensus at any position except 28 ( figure 3a and table 4 ) .
there must be other determinants allowing for or suppressing  formation in these other trnas with a u at 28 .
the uridine to be modified does not have to participate in watson - crick base - pairing but nucleotides participating in base - pairing 3 to the modified position appear to be required to constitute an efficient substrate .
these results correlate well with the observation that , in vivo , yeast pus1p formed  at position 26 in yeast trna(cca ) , where the uridine is in the hinge between the d stem - loop and the asl , and is followed by 5  bp in the asl stem ( 33 ) . in addition , both yeast and mouse pus1p modify the uridine in position 1 on yeast trna(acg ) , with base pairing present in the acceptor stem 3 to the position of modification ( 33 ) . and finally , yeast and mammalian pus1p modifies several uridines in the extensively base - paired asl of yeast pre - trna(uau ) in vitro and in vivo ( 27,29,33 ) . the lack of a strict substrate sequence requirement for hpus1p can be contrasted with the rigid recognition motif of yeast pseudouridine synthase 7 ( pus7p ) , another synthase that can recognize more than one site in trna and other types of rna ( 44 ) . with pus7p
there is an absolute requirement for a u at 2 and an a at + 1 relative to the uridine modified ( 44 ) . of the sequenced yeast and human trnas , all that have the proscribed pus7p recognition sequence ( 17 total )
the other site - specific  synthases modify only one site in trnas ( pseudouridine synthases 4/10 , 6 , 8/9 ) or one small region in many trnas ( pus3p ) ( 48,49,5962 ) . with these latter 
synthases the local trna structure appears to be a determining factor for substrate recognition when one considers the results of trua ( pus3p homolog ) and trub ( pseudouridine synthase 4 homolog ) structural studies ( 46,47,63,64 ) .
given the requirement that the uridine to be modified be in a base - paired stem , why is the uridine at position 44 in yeast u2 snrna modified by pus1p ( 33 ) when that position is not considered to be in a region that is base paired ?
yeast pus1p can modify u2 snrna in vitro and in vivo , but mpus1p only modifies u44 on u2 snrna in vivo , in a pus1 strain ( 33,36 ) .
an alternative structure , perhaps in the ribonucleoprotein particle where u44 is base - paired , may form in vivo .
it is also possible that another rna , such as a snorna , base pairs in the region of u44 and forms a stem that can be recognized by yeast or mouse pus1p .
it is interesting to note that  formation in u2 snrna in c. elegans is not dependent on pus1p activity ( 28 ) .
the results with mutations in the tc stem - loop suggest that there is a minimum size requirement for this loop but the actual sequence , or base pairing in the stem , are not critical .
a minimum size requirement may be a reason that the v stem - loop of trna(uga ) does not substitute for the tc stem - loop when combined with the asl .
the v stem - loop in trna(uga ) is predicted to have only 3  nt in the loop .
the fact that the tc stem - loop is separated from the asl by 4  nt in the mini - substrate and the v stem - loop has no separation , may also be a factor in why the tc stem - loop can participate in creating a mini - substrate that is recognized by hpus1p .
the only  synthase in the trua family that has been crystallized is bacterial trua ( see above ) and the structures were obtained in the presence of substrate trnas ( 46,47 ) .
trua is a dimer and makes contact with the asl ( positions 3840 are modified if a u is present at those sites ) , the d stem , and the  elbow where the d and tc loops interact .
the v loop or stem - loop and the acceptor stem did not interact with trua ( 47 ) . in this report
the d stem - loop of trna(uga ) was found to be dispensable for hpus1p activity , as was the acceptor stem , but the absence of the v stem - loop from the trna does reduce the amount of  formed . in this trna substrate , the tc stem - loop appears to be the more critical component for recognition by hpus1p
. therefore , even though there is considerable homology between trua and hpus1p , the substrate contacts with the protein are probably different .
yeast pus1p has been shown to be a monomer when bound to trna ( 30 ) and it is reasonable to assume the substrate / enzyme interactions will be fundamentally different between trua and pus1p .
crystal structures for any of the pus1p proteins in the presence of trna substrates will go a long way towards reconciling these differences . with what appear to be relatively lax requirements for substrate recognition in trna , what prevents other uridines , such as those at 39 , 43 , 44 , 63 , or 70 in trna ( figure 1a ) from being modified by hpus1p in vitro ?
modifications in the asl ( 26 , 27 , 28 , 30 ) of all natural substrates of pus1p are only found on the 5 side of the stem .
this specificity is most likely due to the presentation of this side of the asl to the enzyme 's active site cleft , in much the same way the opposite side of the stem is presented to trua for modification of positions 3840 ( 47 ) .
this flipping of the orientation of the substrate in the active site cleft could explain why the tc stem - loop is a critical part of the recognition motif for hpus1p rather than the d stem - loop .
these other sites that have uridines in a stem are not recognized because they are not presented in a conformation compatible with placing the uridine in the active site .
additional natural and mini - substrates will need to be studied to refine the parameters of hpus1p substrate recognition to construct a recognition site that can be used to narrow down the possible modification sites in sra , a relatively large , non - trna substrate of pus1p , where most of the actual  residues have not been identified ( 37,38 ) , or to identify additional substrates .
the known substrates of pus1p may just be the tip of the iceberg of actual rna substrates for this enigmatic enzyme .
national institutes of health ( grant number dk074368 - 01 ) ; south carolina honors college at the university of south carolina ( undergraduate research fellowship and thesis preparation grants to b.s.s . ) .
 pseudouridine synthase 1 ( pus1p ) is an unusual site - specific modification enzyme in that it can modify a number of positions in trnas and can recognize several other types of rna . 
 no consensus recognition sequence or structure has been identified for pus1p . 
 human pus1p was used to determine which structural or sequence elements of human trnaser are necessary for pseudouridine (  ) formation at position 28 in the anticodon stem - loop ( asl ) . 
 some point mutations in the asl stem of trnaser had significant effects on the levels of modification and compensatory mutation , to reform the base pair , restored a wild - type level of  formation . 
 deletion analysis showed that the trnaser tc stem - loop was a determinant for modification in the asl . a mini - substrate composed of the asl and tc stem - loop exhibited significant  formation at position 28 and 
 a number of mutants were tested . 
 substantial base pairing in the asl stem ( 3 out of 5  bp ) is required , but the sequence of the tc loop is not required for modification . when all nucleotides in the asl stem other than u28 were changed in a single mutant , but base pairing was retained , a near wild - type level of modification was observed . 
INTRODUCTION
MATERIALS AND METHODS
Isolation of recombinant hPus1p and site directed mutagenesis of tRNA
RNA synthesis, the tritium-release assay and binding assays
RESULTS
DISCUSSION
FUNDING