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At least 199 records · Page 11Linked to original sources
PubMed1989

Interaction of yeast transcription factor IIIC with dimeric Schizosaccharomyces pombe tRNA(Ser)-tRNA(Met) genes.

A unique tRNA(Ser)-tRNA(Met) tandem gene arrangement was characterized previously from Schizosaccharomyces pombe. Three alleles exist in which a tRNA(Ser) gene is separated by 7 base pairs from an initiator tRNA(Met) gene. Promotion of transcription occurs only within the tRNA(Ser) gene, yielding a dimeric precursor transcript. Using nuclease protection and gel retardation assays, we have analyzed how the Saccharomyces cerevisiae RNA polymerase III transcription factor C (TFIIIC) interacts with this dimeric gene template. The primary interaction site of TFIIIC with the tRNA(Ser) gene is at the 3'-internal control region (ICR), which can be distinguished kinetically from its weaker interaction with the 5'-ICR of the gene. We examined a variety of point mutations and double mutations within the tRNA(Ser) gene which reduce transcription. We found that changes in highly conserved nucleotides within the ICRs reduce TFIIIC binding up to 7-fold compared with the parent suppressor gene. The interaction of TFIIIC with the tRNA(Ser) gene does not sterically prevent stable binding of TFIIIC to the 3'-ICR of the tRNA(Met) gene. However, the affinity of binding of TFIIIC to the dimeric template is 7-fold higher than to the tRNA(Met) gene, alone, demonstrating that the tRNA(Met) gene contains intrinsically weak promoter elements. This may contribute to the inability of the tRNA(Met) gene to independently direct transcription from its ICR elements.

Alleles↗
PubMed1986

Contact areas of the turnip yellow mosaic virus tRNA-like structure interacting with yeast valyl-tRNA synthetase.

The tRNA-like structure of turnip yellow mosaic virus is known to be efficiently recognized and aminoacylated by valyl-tRNA synthetase. The present work reports domains in the isolated tRNA-like fragment (159 terminal nucleotides at the 3'-end of the two viral RNAs) in contact with purified yeast valyl-tRNA synthetase. These domains were determined in protection experiments using chemical and enzymatic structural probes. In addition, new data, re-enforcing the validity of the tertiary folding model for the native RNA, are given. In particular, at the level of the amino acid accepting arm it was found that the two phosphate groups flanking the three guanine residues of loop I are inaccessible to ethylnitrosourea. This is in agreement with a higher-order structure of this loop involving "pseudo knotting", as proposed by Rietveld et al. (1982). Valyl-tRNA synthetase efficiently protects the viral RNA against digestion by single-strand-specific S1 nuclease at the level of the anticodon loop. With cobra venom ribonuclease, specific for double-stranded regions of RNA, protection was detected on both sides of the anticodon arm and at the 5'-ends of loop I, a region that is involved in the building up of the acceptor arm. Loop II, which is topologically homologous to the T-loop of canonical tRNA was likewise protected. Weak protection was observed between arms I and II, and at the 3'-side of arm V. This arm, located at the 5'-side of arm IV (homologous to the D-arm of tRNA), does not participate in the pseudo-knotted model of the valine acceptor arm. Ethylnitrosourea was used to determine the phosphates of the tRNA-like structure in close contact with the synthetase. These are grouped in several stretches scattered over the RNA molecule. In agreement with the nuclease digestion results, protected phosphates are located in arms I, II, and III. Additionally, this chemical probe permits detection of other protected phosphates on the 3'-side of arm IV and on both sides of arm V. When displayed in the three-dimensional model of the tRNA-like structure, protected areas are localized on both limbs of the L-shaped RNA. It appears that valyl-tRNA synthetase embraces the entire tRNA-like structure. This is reminiscent of the interaction model of canonical yeast tRNAVal with its cognate synthetase.

Alkylation↗
PubMed1987

Covalent coupling of the variable loop of the elongator methionine tRNA to a specific lysine residue in Escherichia coli methionyl-tRNA synthetase.

A lysine-reactive cross-linker has been coupled to the minor base 3-(3-amino-3-carboxypropyl)uridine in the variable loop of the Escherichia coli elongator methionine tRNA (tRNA(mMet]. Incubation of the derivatized tRNA with E. coli methionyl-tRNA synthetase (MetRS) resulted in covalent coupling of the protein and nucleic acid and loss of amino acid acceptor activity of the enzyme. One mole of tRNA was cross-linked per mole of enzyme inactivated. Enzyme activity was largely restored by release of the bound tRNA following cleavage of the disulfide bond in the cross-linker with a sulfhydryl reagent. The cross-linking reaction was effectively inhibited by unmodified tRNA(mMet) but not by noncognate tRNA(Phe). The covalent complex was digested with trypsin, and the resulting tRNA-bound peptides were isolated by anion-exchange chromatography. The cross-linked peptides were released from the tRNA by cleavage in the disulfide bond of the cross-linker and purified by reverse-phase high-pressure liquid chromatography, yielding one major peptide plus several minor peptides. Amino acid analysis indicated that the major product was an octadecapeptide cross-linked to tRNA(mMet) through lysine residue 596 in the primary sequence of MetRS. The N-terminal sequence of the peptide was determined to be Val-Ala-Leu-Ile-Glu-Asn-Ala-Glu-Phe-Val, corresponding to residues 582-591 in MetRS. The procedures described here should be applicable to the determination of peptide sequences near the variable loop of other tRNAs containing the 3-(3-amino-3-carboxypropyl)uracil base when such tRNAs are bound to specific proteins.

Amino Acid Sequence↗
PubMed2003

Non-canonical eukaryotic glutaminyl- and glutamyl-tRNA synthetases form mitochondrial aminoacyl-tRNA in Trypanosoma brucei.

Glutaminyl-tRNA synthetase is thought to be absent from organelles. Instead, Gln-tRNA is formed via the transamidation pathway, the other route to this essential compound in protein biosynthesis. However, it was previously shown that glutaminyl-tRNA synthetase activity is present in Leishmania mitochondria. This work identifies genes encoding glutaminyl- and glutamyl-tRNA synthetase in the closely related organism Trypanosoma brucei. Down-regulation of their respective gene products by RNA interference showed that (i) they are essential for the growth of insect stage T. brucei and (ii) they are responsible for essentially all of the glutaminyl- and glutamyl-tRNA synthetase activity detected in both the cytosol and the mitochondria. In vitro aminoacylation experiments with the recombinant T. brucei enzymes and total tRNA confirmed the identity of the two aminoacyl-tRNA synthetases. Interestingly, T. brucei uses the same eukaryotic-type glutaminyl-tRNA synthetase to form mitochondrial and cytosolic Gln-tRNA. The formation of Glu-tRNA in mitochondria and the cytoplasm is catalyzed by a single eukaryotic-type discriminating glutamyl-tRNA synthetase. T. brucei, similar to Leishmania, imports all of its mitochondrial tRNAs from the cytosol. The use of these two eukaryotic-type enzymes in mitochondria may therefore reflect an adaptation to the situation in which the cytosol and mitochondria use the same set of tRNAs.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Macromolecular complex of aminoacyl-tRNA synthetases from sheep liver. Identification of the methionyl-tRNA synthetase component by affinity labeling.

Both the tRNA aminoacylation and amino-acid-dependent ATP-PPi exchange activities of monomeric trypsin-modified methionyl-tRNA synthetase from sheep liver are lost upon incubation with oxidized initiator tRNAMet. The inactivation, which reflects the formation of a Schiff's base between the 5'-terminal adenosine of tRNA and a lysine within the catalytic site of the enzyme, is accompanied by the covalent attachment of one tRNA molecule per enzyme molecule. The affinity labeling method is applied to the sheep liver complex of Mr 10(6) carrying seven aminoacyl-tRNA synthetase activities, from which the monomeric trypsin-modified methionyl-tRNA synthetase (Mr 68 000) was derived. Upon incubation with oxidized initiator tRNAMet, the methionyl-tRNA synthetase activity of the complex is lost. Of the eleven polypeptide chains composing the high-molecular-weight complex, only one polypeptide chain with Mr 103 000 reacts with the modified tRNAMet. The blocking by periodate-treated tRNA of the methionyl-tRNA synthetase activity in the complex has no effect on the other aminoacyl-tRNA synthetase activities. This strongly argues in favor of the independent parallel functioning of the seven aminoacyl-tRNA synthetases associated in a high-molecular-weight complex.

Affinity Labels↗
PubMed2006

The La protein functions redundantly with tRNA modification enzymes to ensure tRNA structural stability.

Although the La protein stabilizes nascent pre-tRNAs from nucleases, influences the pathway of pre-tRNA maturation, and assists correct folding of certain pre-tRNAs, it is dispensable for growth in both budding and fission yeast. Here we show that the Saccharomyces cerevisiae La shares functional redundancy with both tRNA modification enzymes and other proteins that contact tRNAs during their biogenesis. La is important for growth in the presence of mutations in either the arginyl tRNA synthetase or the tRNA modification enzyme Trm1p. In addition, two pseudouridine synthases, PUS3 and PUS4, are important for growth in strains carrying a mutation in tRNA(Arg)(CCG) and are essential when La is deleted in these strains. Depletion of Pus3p results in accumulation of the aminoacylated mutant tRNA(Arg)(CCG) in nuclei, while depletion of Pus4p results in decreased stability of the mutant tRNA. Interestingly, the degradation of mutant unstable forms of tRNA(Arg)(CCG) does not require the Trf4p poly(A) polymerase, suggesting that yeast cells possess multiple pathways for tRNA decay. These data demonstrate that La functions redundantly with both tRNA modifications and proteins that associate with tRNAs to achieve tRNA structural stability and efficient biogenesis.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

[Specific modification of phenylalanine:tRNA-ligases of E. coli MRE-600 with N-chlorambucilyl-14c-phenylalanyl-tRNA].

N-Chlorambucilyl-[14C]phenylalanyl-tRNA was used for the affinity modification of phenylalanine : tRNA-ligase from E. coli MRE-600. It has been found that N-chlorambucilyl-[14C]phenylalanyl-tRNA selectively inactivates phenylalanine : tRNA-lagase that results in formation of a covalent bond between the tRNA derivative and the enzyme at pH 5.8, 25 degrees C. The rate fall of the aminoacylation of tRNA with [14C]phenylalanine was observed after the enzyme incubation with N-chlorambucilyl-[14C]phenylalanyl-tRNA at pH 7.5, 25 degrees C. It has been shown that this modification results in a similar rate decrease of tRNA aminoacylation with [14C]phenylalanine, ATP-[32P]pyrophosphate exchange and reaction of the enzymatic deacylation of [14C]phenylalanyl-tRNA. This fact evidences in favour of the possibility of the alkylation to proceed in the proximity of the active centre of the enzyme. The covalent complex obtained seems to be an interesting model for the studies of the mechanisms involved in tRNA aminoacylation as well as for elucidation of the tertiary structure of tRNA bound with the enzyme.

Adenosine Triphosphate↗
PubMed1993

Identity determinants of human tRNA(Ser): sequence elements necessary for serylation and maturation of a tRNA with a long extra arm.

Recently, there has been much progress in understanding tRNA identity, i.e. in elucidating the sets of nucleotides that are responsible for the specific aminoacylation of a tRNA with its cognate amino acid. Interest focused, however, on tRNAs from Escherichia coli and yeast. Here we have identified the major and minor determinants of human tRNA(Ser) which were revealed by an identity switch from human tRNA(Val) to tRNA(Ser). We used in vitro transcripts and subsequent aminoacylation by HeLa S100 extract to determine the kinetic parameter Vmax/Km. The two major identity elements which are absolutely required for aminoacylation by human seryl-tRNA synthetase are the discriminator base and the long extra arm. This is in contrast to E. coli tRNA(Ser) where the discriminator base is unimportant, whereas identity determinants in the acceptor stem are required. Other sequence elements have an influence not only on serylation, but also on tRNA maturation in vitro, i.e. on pre-tRNA processing and base modification. These nucleotides are located in the DHU and the T phi C arm and are probably necessary for the proper folding of tRNAs containing a long extra arm. A34 to inosine modification depends highly on the correct three-dimensional structure of the tRNA, whereas A58 to m1A methylation does not rely on the three-dimensional folding of the substrate. This is the first tRNA identity switch involving the exchange of a short versus a long extra arm.

Acylation↗
PubMed2001

A dual-specific Glu-tRNA(Gln) and Asp-tRNA(Asn) amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans.

The gatC, gatA and gatB genes encoding the three subunits of glutamyl-tRNA(Gln) amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon-like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu-tRNA(Gln) to Gln-tRNA(Gln) and Asp-tRNA(Asn) to Asn-tRNA(Asn). Biochemical analysis revealed that neither glutaminyl-tRNA synthetase nor asparaginyl-tRNA synthetase is present in A. ferrooxidans, but that glutamyl-tRNA synthetase and aspartyl-tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln-tRNA and Asn-tRNA in A. ferrooxidans.

Asparagine↗
PubMed1998

tRNA(Pro) anticodon recognition by Thermus thermophilus prolyl-tRNA synthetase.

BACKGROUND: Most aminoacyl-tRNA synthetases (aaRSs) specifically recognize all or part of the anticodon triplet of nucleotides of their cognate tRNAs. Class IIa and class IIb aaRSs possess structurally distinct tRNA anticodon-binding domains. The class IIb enzymes (LysRS, AspRS and AsnRS) have an N-terminal beta-barrel domain (OB-fold); the interactions of this domain with the anticodon stem-loop are structurally well characterised for AspRS and LysRS. Four out of five class IIa enzymes (ProRS, ThrRS, HisRS and GlyRS, but not SerRS) have a C-terminal anticodon-binding domain with an alpha/beta fold, not yet found in any other protein. The mode of RNA binding by this domain is hitherto unknown as is the rationale, if any, behind classification of anticodon-binding domains for different aaRSs. RESULTS: The crystal structure of Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) in complex with tRNA(Pro) has been determined at 3.5 A resolution by molecular replacement using the native enzyme structure. One tRNA molecule, of which only the lower two-thirds is well ordered, is found bound to the synthetase dimer. The C-terminal anticodon-binding domain binds to the anticodon stem-loop from the major groove side. Binding to tRNA by ProRSTT is reminiscent of the interaction of class IIb enzymes with cognate tRNAs, but only three of the anticodon-loop bases become splayed out (bases 35-37) rather than five (bases 33-37) in the case of class IIb enzymes. The two anticodon bases conserved in all tRNA(Pro), G35 and G36, are specifically recognised by ProRSTT. CONCLUSIONS: For the synthetases possessing the class IIa anticodon-binding domain (ProRS, ThrRS and GlyRS, with the exception of HisRS), the two anticodon bases 35 and 36 are sufficient to uniquely identify the cognate tRNA (GG for proline, GU for threonine, CC for glycine), because these amino acids occupy full codon groups. The structure of ProRSTT in complex with its cognate tRNA shows that these two bases specifically interact with the enzyme, whereas base 34, which can be any base, is stacked under base 33 and makes no interactions with the synthetase. This is in agreement with biochemical experiments which identify bases 35 and 36 as major tRNA identity elements. In contrast, class IIb synthetases (AspRS, AsnRS and LysRS) have a distinct anticodon-binding domain that specifically recognises all three anticodon bases. This again correlates with the requirements of the genetic code for cognate tRNA identification, as the class IIb amino acids occupy half codon groups.

Amino Acid Sequence↗
PubMed2002

Dual function of the tRNA(m(5)U54)methyltransferase in tRNA maturation.

A 5-methyluridine (m(5)U) residue at position 54 is a conserved feature of bacterial and eukaryotic tRNAs. The methylation of U54 is catalyzed by the tRNA(m5U54)methyltransferase, which in Saccharomyces cerevisiae is encoded by the nonessential TRM2 gene. In this study, we identified four different strains with mutant forms of tRNA(Ser)CGA. The absence of the TRM2 gene in these strains decreased the stability of tRNA(Ser)CGA and induced lethality. Two alleles of TRM2 encoding catalytically inactive tRNA(m5U54)methyltransferases were able to stabilize tRNA(Ser)CGA in one of the mutants, revealing a role for the Trm2 protein per se in tRNA maturation. Other tRNA modification enzymes interacting with tRNA(Ser)CGA in the maturation process, such as Pus4p, Trm1 p, and Trm3p were essential or important for growth of the tRNA(Ser)CGA mutants. Moreover, Lhp1p, a protein binding RNA polymerase III transcripts, was required to stabilize the mutant tRNAs. Based on our results, we suggest that tRNA modification enzymes might have a role in tRNA maturation not necessarily linked to their known catalytic activity.

Antigens, Viral↗
PubMed1998

Overproduction of selenocysteine tRNA in Chinese hamster ovary cells following transfection of the mouse tRNA[Ser]Sec gene.

Selenocysteine insertion during selenoprotein biosynthesis begins with the aminoacylation of selenocysteine tRNA[ser]sec with serine, the conversion of the serine moiety to selenocysteine, and the recognition of specific UGA codons within the mRNA. Selenocysteine tRNA[ser]sec exists as two major forms, differing by methylation of the ribose portion of the nucleotide at the wobble position of the anticodon. The levels and relative distribution of these two forms of the tRNA are influenced by selenium in mammalian cells and tissues. We have generated Chinese hamster ovary cells that exhibit increased levels of tRNA[ser]sec following transfection of the mouse tRNA[ser]sec gene. The levels of selenocysteine tRNA[ser]sec in transfectants increased proportionally to the number of stably integrated copies of the tRNA[ser]sec gene. Although we were able to generate transfectants overproducing tRNA[ser]sec by as much as tenfold, the additional tRNA was principally retained in the unmethylated form. Selenium supplementation could not significantly affect the relative distributions of the two major selenocysteine tRNA[ser]sec isoacceptors. In addition, increased levels of tRNA[ser]sec did not result in measurable alterations in the levels of selenoproteins, including glutathione peroxidase.

Amino Acyl-tRNA Synthetases↗
PubMed1994

Crystal structure of unmodified tRNA(Gln) complexed with glutaminyl-tRNA synthetase and ATP suggests a possible role for pseudo-uridines in stabilization of RNA structure.

tRNA(2Gln) made in vitro by transcription with T7 RNA polymerase does not contain the pseudouridines at positions 38, 39, and 55, the 4-thiouridine at position 8, or any of the methylated bases found in the tRNA(2Gln) made in vivo. Cocrystals of unmodified tRNA(2Gln) complexed with glutaminyl-tRNA synthetase from Escherichia coli are isomorphous with those of the complex with modified tRNA(2Gln). A difference electron density map between the complexes with modified and unmodified tRNAs calculated at 2.5-A resolution shows no differences in the protein or tRNA structures, except for some very small shifts in atoms contacting the thiol at the 4 position of uridine 8 that are required to accommodate the smaller oxygen in the unmodified tRNA. Perhaps the most functionally significant change in the unmodified tRNA is the absence of the specifically bound water molecules that are observed to cross-link the N5 of the pseudo-uridines to their 5' phosphate. This suggests a possible role for pseudouridinylation in stabilization of the tRNA through water-mediated linking of these modified bases to the backbone, which is consistent with the lower thermal stability of the unmodified tRNA. An identical water-bridging structure is possible at four of the five other psuedo-uridines in known tRNA structures.

Adenosine Triphosphate↗
PubMed2003

Prokaryotic and eukaryotic tetrameric phenylalanyl-tRNA synthetases display conservation of the binding mode of the tRNA(Phe) CCA end.

The interaction of human phenylalanyl-tRNA synthetase, a eukaryotic prototype with an unknown three-dimensional structure, with the tRNA(Phe) acceptor end was studied by s(4)U-induced affinity cross-linking with human tRNA(Phe) derivatives site-specifically substituted at the single-stranded 3' end. Two different subunits of the enzyme bind two adjacent nucleotides of the tRNA(Phe) 3' end: nucleotide 76 is associated with the catalytic alpha subunit, while nucleotide 75 is in contact with the beta subunit. The binding mode is similar to that revealed previously in structural and affinity cross-linking studies of the prokaryotic Thermus thermophilus phenylalanyl-tRNA synthetase. Our results suggest that the distinctive features of tRNA(Phe) acceptor end binding are conserved for the eukaryotic and prokaryotic tetrameric phenylalanyl-tRNA synthetases despite their significant differences in the domain composition of the beta subunits. The data from affinity cross-linking experiments with human phenylalanyl-tRNA synthetase complexed with small ligands (ATP and/or phenylalanine or a stable synthetic analogue of phenylalanyl adenylate) reveal that the location of the tRNA(Phe) acceptor end varies with the presence and nature of other substrates. The lack of substrate activity of human tRNA(Phe) substituted with s(4)U at the 3'-terminal position suggests that base-specific interactions of the terminal adenosine are critically important for a productive interaction. The conformational rearrangement of the tRNA 3' end induced by the other substrates and dictated by base-specific contacts of the terminal nucleotide is an additional means of ensuring the phenylalanylation specificity in both prokaryotic and eukaryotic systems.

Adenosine Monophosphate↗
PubMed2003

Limited set of amino acid residues in a class Ia aminoacyl-tRNA synthetase is crucial for tRNA binding.

The aim of this work was to characterize crucial amino acids for the aminoacylation of tRNA(Arg) by yeast arginyl-tRNA synthetase. Alanine mutagenesis was used to probe all the side chain mediated interactions that occur between tRNA(Arg2)(ICG) and ArgRS. The effects of the substitutions were analyzed in vivo in an ArgRS-knockout strain and in vitro by measuring the aminoacylation efficiencies for two distinct tRNA(Arg) isoacceptors. Nine mutants that generate lethal phenotypes were identified, suggesting that only a limited set of side chain mediated interactions is essential for tRNA recognition. The majority of the lethal mutants was mapped to the anticodon binding domain of ArgRS, a helix bundle that is characteristic for class Ia synthetases. The alanine mutations induce drastic decreases in the tRNA charging rates, which is correlated with a loss in affinity in the catalytic site for ATP. One of those lethal mutations corresponds to an Arg residue that is strictly conserved in all class Ia synthetases. In the known crystallographic structures of complexes of tRNAs and class Ia synthetases, this invariant Arg residue stabilizes the idiosyncratic conformation of the anticodon loop. This paper also highlights the crucial role of the tRNA and enzyme plasticity upon binding. Divalent ions are also shown to contribute to the induced fit process as they may stabilize the local tRNA-enzyme interface. Furthermore, one lethal phenotype can be reverted in the presence of high Mg(2+) concentrations. In contrast with the bacterial system, in yeast arginyl-tRNA synthetase, no lethal mutation has been found in the ArgRS specific domain recognizing the Dhu-loop of the tRNA(Arg). Mutations in this domain have no effects on tRNA(Arg) aminoacylation, thus confirming that Saccharomyces cerevisiae and other fungi belong to a distinct class of ArgRS.

Acylation↗
PubMed2006

Phenylalanyl-tRNA synthetase contains a dispensable RNA-binding domain that contributes to the editing of noncognate aminoacyl-tRNA.

Phenylalanyl-tRNA synthetase (PheRS) is a multidomain (alphabeta)2 heterotetrameric protein responsible for synthesizing Phe-tRNA(Phe) during protein synthesis. Previous studies showed that the alpha subunit forms the catalytic core of the enzyme, while the beta subunit contains a number of autonomous structural modules with a wide range of functions including tRNA anticodon binding and editing of the misaminoacylated species Tyr-tRNA(Phe). The B2 domain of the beta subunit is a structural homologue of the EMAPII/OB fold, which has been shown in other systems to contribute to tRNA binding. Structural studies of PheRS indicated that the B2 domain is distant from bound tRNA(Phe), leaving the role of this module in question. On the basis of homology modeling with other EMAPII domain-containing proteins, the 110 amino acid B2 domain was deleted to produce PheRS deltaB2. Full-length PheRS and PheRS deltaB2 showed comparable kinetics for in vitro aminoacylation, and both enzymes complemented a defect in phenylalanylation in vivo. PheRS deltaB2 showed a 2-fold drop compared to full-length PheRS in the catalytic efficiency (kcat/KM) of Tyr-tRNA(Phe) hydrolysis, suggesting a role for the B2 domain in post-transfer editing. A comparison of tRNA binding by full-length PheRS and PheRS deltaB2 indicated that the B2 domain acts as a secondary tRNA-binding site that could contribute to editing by promoting the translocation of mischarged tRNA to the editing site of PheRS. This proposed role for the B2 domain of PheRS is consistent with previous studies, suggesting that the highly conserved EMAPII fold is able to modulate the affinity of tRNA for its primary binding site.

Amino Acid Sequence↗
PubMed1996

Conservation in evolution for a small monomeric phenylalanyl-tRNA synthetase of the tRNA(Phe) recognition nucleotides and initial aminoacylation site.

We previously showed that yeast mitochondrial phenylalanyl-tRNA synthetase (MSF protein) is evolutionarily distant to the cytoplasmic counterpart based on a high degree of divergence in protein sequence, molecular mass, and quaternary structure. Using yeast cytoplasmic tRNA(Phe) which is efficiently aminoacylated by MSF protein, we report here the tRNA(Phe) primary site of aminoacylation and the identity determinants for MSF protein. As for the cytoplasmic phenylalanyl-tRNA synthetase (Sampson, J. R., Di Renzo, A. B., Behlen, L. S., & Uhlenbeck, O. C. (1989) Science 243, 1363-1366), MSF protein recognizes nucleotides from the anticodon and the acceptor end including base A73 and, as shown here, adjacent G1-C72 base pair or at least C72 base. This indicates that the way of tRNA(Phe) binding for the two phenylalanine enzymes is conserved in evolution. However, tRNA(Phe) tertiary structure seems more critical for the interaction with the cytoplasmic enzyme than with MSF protein, and unlike cytoplasmic phenylalanyl-tRNA synthetase, the small size of the monomeric MSF protein probably does not allow contacts with residue 20 at the top corner of the L molecule. We also show that MSF protein preferentially aminoacylates the terminal 2'-OH group of tRNA(Phe) but with a catalytic efficiency for tRNA(Phe)-CC-3'-deoxyadenosine reduced 100-fold from that of native tRNA(Phe), suggesting a role of the terminal 3'-OH in catalysis. The loss is only 1.5-fold when tRNA(Phe)-CC-3'-deoxyadenosine is aminoacylated by yeast cytoplasmic PheRS (Sprinzl, M., & Cramer, F. (1973) Nature 245, 3-5), indicating mechanistic differences between the two PheRS's active sites for the amino acid transfer step.

Amino Acid Sequence↗
PubMed1999

tRNA discrimination at the binding step by a class II aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic information by accurately joining amino acids to their cognate transfer RNAs. Here, tRNA discrimination at the level of binding by Escherichia coli histidyl-tRNA synthetase is addressed by filter binding, analytical ultracentrifugation, and iodine footprinting experiments. Competitive filter binding assays show that the presence of an adenylate analogue 5'-O-[N-(L-histidyl)sulfamoyl]adenosine, HSA, decreased the apparent dissociation constant (K(D)) for cognate tRNA(His) by more than 3-fold (from 3.87 to 1.17 microM), and doubled the apparent K(D) for noncognate tRNA(Phe) (from 7.3 to 14.5 microM). By contrast, no binding discrimination against mutant U73 tRNA(His) was observed, even in the presence of HSA. Additional filter binding studies showed tighter binding of both cognate and noncognate tRNAs by G405D mutant HisRS [Yan, W., Augustine, J., and Francklyn, C. (1996) Biochemistry 35, 6559], which possesses a single amino acid change in the C-terminal anticodon binding domain. Discrimination against noncognate tRNA was also observed in sedimentation velocity experiments, which showed that a stable complex was formed with the cognate tRNA(His) but not with noncognate tRNA(Phe). Footprinting experiments on wild-type versus G405D HisRS revealed characteristic alterations in the pattern of protection and enhancement of iodine cleavage at phosphates 5' to tRNA nucleotides in the anticodon and hinge regions. Together, these results suggest that the anticodon and core regions play major roles in the initial binding discrimination between cognate and noncognate tRNAs, whereas acceptor stem nucleotides, particularly at position 73, influence the reaction at steps after binding of tRNA.

Adenosine↗