Studies on valyl-tRNA synthetase and tRNA from Escherichia coli. II. Interaction between valyl-tRNA synthetase and valine acceptor tRNA.
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Adenine Nucleotides↗
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Genomic DNA sequences of mitochondrial tRNA genes in fungi Penicillium chrysogenum KCTC1262: tRNA(arg), tRNA(asn) and tRNA(tyr) genes.
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Base Sequence↗
Isolation and characterization of 2-methyladenosine from Escherichia coli tRNA Glu 2 , tRNA Asp 1 , tRNA His 1 and tRNA Arg .
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Adenosine↗
A yeast tRNA(Arg) gene can act as promoter for a 5' flank deficient, non-transcribable tRNA(SUP)6 gene to produce biologically active suppressor tRNA.
In S. cerevisiae most tRNA genes are located and expressed as single entities. The tDNA(Arg)-tDNA(Asp) pair, however, is transcribed into a dimeric precursor before being processed into two mature tRNA species. The second gene of this pair, tDNA(Asp), is totally dependent on the first gene, tDNA(Arg), and its promoter components, for homologous in vitro transcription. The second gene in the pair is now replaced by the ochre suppressor tDNA(SUP)6-o, which, by itself, cannot be transcribed because of a nonfunctional 5' flanking region. The tDNA(Arg)-tDNA(SUP)6-o was transcribed into a dimeric precursor which was processed to mature tRNA molecules as judged in vitro by electrophoretic separation, and in vivo by their ability to suppress ochre but not amber yeast mutations. Mutations in the internal promoter of the first gene decreased transcription, both in vitro and in vivo, of the second-tRNA(SUP)6-o-gene. Thus tDNA(Arg) with its 5' flanking region can act as an external promoter for other RNA polymerase III-read genes that are by themselves inactive due to impaired promoter/modulator regions.
Base Sequence↗
Regulation of elongation factor G GTPase activity by the ribosomal state. The effects of initiation factors and differentially bound tRNA, aminoacyl-tRNA, and peptidyl-tRNA.
The elongation factor G (EF-G) is responsible for the translocation of the ribosome along the mRNA chain. Under in vitro conditions, EF-G exhibits a very active uncoupled GTPase activity which is dependent on the presence of ribosomes and is modulated by mRNA-dependent binding of tRNA. In the absence of tRNA, uncoupled EF-G GTPase is inhibited by initiation factors IF1 and IF3, but not by initiation factor IF2. In the presence of N-fMet-tRNAfMet and poly(A,U,G) or in the presence of N-acetyl-Phe-tRNAPhe and poly(U), initiation factor IF2 causes an additional decrease of the uncoupled EF-G GTPase activity. This effect, however, is dependent on the presence of IF1 and IF3 and is obviously due to the mRNA- and initiation factor-dependent binding of N-fMet-tRNAfMet and N-acetyl-Phe-tRNAPhe, respectively, to the ribosomal P-site. Non-enzymatic binding of N-fMet-tRNAfMet and N-acetyl-Phe-tRNAPhe, however, causes a stimulation of uncoupled EF-G GTPase activity. The same effects are observed for Met-tRNA, Phe-tRNAPhe and uncharged tRNA. These findings are discussed in the light of the three-site model of the ribosome and the mechanism of translocation.
Chromatography, DEAE-Cellulose↗
Mirror image alternative interaction patterns of the same tRNA with either class I arginyl-tRNA synthetase or class II aspartyl-tRNA synthetase.
Gene cloning, overproduction and an efficient purification protocol of yeast arginyl-tRNA synthetase (ArgRS) as well as the interaction patterns of this protein with cognate tRNAArgand non-cognate tRNAAspare described. This work was motivated by the fact that the in vitro transcript of tRNAAspis of dual aminoacylation specificity and is not only aspartylated but also efficiently arginylated. The crystal structure of the complex between class II aspartyl-tRNA synthetase (AspRS) and tRNAAsp, as well as early biochemical data, have shown that tRNAAspis recognized by its variable region side. Here we show by footprinting with enzymatic and chemical probes that transcribed tRNAAspis contacted by class I ArgRS along the opposite D arm side, as is homologous tRNAArg, but with idiosyncratic interaction patterns. Besides protection, footprints also show enhanced accessibility of the tRNAs to the structural probes, indicative of conformational changes in the complexed tRNAs. These different patterns are interpreted in relation to the alternative arginine identity sets found in the anticodon loops of tRNAArgand tRNAAsp. The mirror image alternative interaction patterns of unmodified tRNAAspwith either class I ArgRS or class II AspRS, accounting for the dual identity of this tRNA, are discussed in relation to the class defining features of the synthetases. This study indicates that complex formation between unmodified tRNAAspand either ArgRS and AspRS is solely governed by the proteins.
Anticodon↗
Phenylalanyl-tRNA synthetase from yeast and its discrimination of 19 amino acids in aminoacylation of tRNA(Phe)-C-C-A and tRNA(Phe)-C-C-A(3'NH2).
For discrimination between phenylalanine and 18 other naturally occurring non-cognate amino acids by the class II aminoacyl-tRNA synthetase specific for phenylalanine, discrimination factors, D, of 190-6300 have been determined from kcal and K(m) values. Generally, phenylalanyl-tRNA synthetase is more specific than the class II enzymes specific for Lys and Thr, but works with lower accuracy than the class I enzymes specific for IIe, Tyr, and Arg. In aminoacylation of tRNA(Phe)-C-C-A(3'NH2) discrimination factors D1 vary between 80-1610. Pre-transfer proof-reading factors II1 are in the range 2.3-74, post-transfer proof-reading factors II2 in the range 1.0-4.6, showing that pre-transfer proof-reading is the main correction step, post-transfer proofreading is less effective or negligible. Initial discrimination factors (I1 and I2) caused by differences in Gibbs free energies of binding between phenylalanine and non-cognate amino acids have been calculated assuming a two-step binding process. Factors I1 can be related to hydrophobic-interaction forces depending on accessible surface areas of the amino acids, factors I2 scatter about a low mean value and do not show any relation to amino acid structures or surfaces, indicating less checking of amino acid side chains in the putative second binding step.
Acylation↗
Novel mechanism of post-transcriptional modification of tRNA. Insertion of bases of Q precursors into tRNA by a specific tRNA transglycosylase reaction.
The guanine insertion enzyme isolated from Escherichia coli (tRNA transglycosylase) catalyzed the incorporation of bases of Q (queuosine) precursors into E. coli undermodified tRNAAsn and tRNATyr. These bases of Q precursors were inserted in the first position of the anticodon of tRNASn and tRNATyr, replacing guanine originally located in that position. This is a novel type of post-transcriptional modification, inserting a modified base into the polynucleotide chain by cleavage of the N--C glycoside bond without breakage of the phosphodiester bond. One of the bases of Q precursors, 7-(aminomethyl)-7-deazaguanine, was found in the acid-soluble fraction of E. coli cells, supporting the conclusion that formation of Q, 7-(3,4-trans-4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanosine, in tRNA in vivo actually proceeds by the tRNA transglycosylase reaction.
Escherichia coli↗
New photoactivatable structural and affinity probes of RNAs: specific features and applications for mapping of spermine binding sites in yeast tRNA(Asp) and interaction of this tRNA with yeast aspartyl-tRNA synthetase.
Aryldiazonium salts are shown to be useful as phototriggered structural probes for RNA mapping as well as for footprinting of RNA/protein interaction. In particular the yeast tRNA(Asp)/aspartyl-tRNA synthetase complex is shown to involve the variable loop face and the concave side of the L-shaped nucleic acid bound to a lipophilic area of the enzyme. When chemically linked to spermine, the photoactive group cleaves RNA at polyamine binding sites; 3-4 spermines have been located in the tRNA(Asp), stabilizing the central part of the molecule in regions where two ribose-phosphate strands are close to each other.
Affinity Labels↗
Conformational flexibility of tRNA: structural changes in yeast tRNA(Asp) upon binding to aspartyl-tRNA synthetase.
The availability of several X-ray structures at atomic resolution of tRNA(Asp) from yeast, both in its free state and complexed with its cognate tRNA-synthetase, enables a detailed examination of the conformational changes due to interaction with the enzyme. Although the molecule conserves its general L shape, its conformation undergoes important modifications. They may be described as a bending of the two arms which brings the 3' acceptor end and the anticodon part closer together, completed by a drastic change of the anticodon loop, which puts the anticodon bases in a more exposed position, facilitating their interaction with the synthetase. The packing interactions in the crystals are also discussed. Finally, the results of protection studies by chemical probes in solution are discussed in view of the RNA-protein contacts observed in the crystals.
Aspartate-tRNA Ligase↗
Responsibility of tRNA(Ile) for spermine stimulation of rat liver Ile-tRNA formation.
To determine whether tRNA or aminoacyl-tRNA synthetase is responsible for spermine stimulation of rat liver Ile-tRNA formation, homologous and heterologous Ile-tRNA formations were carried out with Escherichia coli and rat liver tRNA(Ile) and their respective purified Ile-tRNA synthetases. Spermine stimulation was observed only when tRNA from the rat liver was used. Spermine bound to rat liver tRNA(Ile) but not to the purified aminoacyl-tRNA synthetase complex. Kinetic analysis of Ile-tRNA formation revealed that spermine increased the Vmax and Km values for rat liver tRNA(Ile). The Km value for ATP and isoleucine did not change significantly in the presence of spermine. Furthermore, higher concentrations of rat liver tRNA(Ile) tended to inhibit Ile-tRNA formation if spermine was absent. Spermine restored isoleucine-dependent PPi-ATP exchange in the presence of rat liver tRNA(Ile), an inhibitor of this exchange. The nucleotide sequence of rat liver tRNA(Ile) was determined and compared with that of E. coli tRNA(Ile). Differences in nucleotide sequences of the two tRNAs(Ile) were observed mainly in the acceptor and anticodon stems. Limited ribonuclease V1 digestion of the 3'-32P-labeled rat liver tRNA(Ile) showed that both the anticodon and acceptor stems were structurally changed by spermine, and that the structural change by spermine was different from that by Mg2+. The influence of spermine on the ribonuclease V1 digestion of E. coli tRNA(Ile) was different from that of rat liver tRNA(Ile). The results suggest that the interaction of spermine with the acceptor and anticodon stems may be important for spermine stimulation of rat liver Ile-tRNA formation.
Amino Acyl-tRNA Synthetases↗
Phenylalanyl-tRNA synthetase of Escherichia coli K 10. Multiple enzyme-aminoacyl-tRNA complexes as a consequence of substrate specificity.
The interaction between Phe-tRNA(Phe) or other acyl-tRNA derivatives thereof and phenylalanyl-tRNA synthetase of Escherichia coli K 10 has been investigated by nonequilibrium dialysis, by fluorescence titration in the presence of 2-p-toluidinylnaphthalene-6-sulfonate, by the kinetics of the aminoacylation of tRNA(Phe), and by the kinetics of the catalytic hydrolysis of Phe-tRNA(Phe). Phe-tRNA(Phe), or derivatives thereof, forms two types of complexes with the synthetase. One type involves the attachment of the phenylalanyl moiety to the phenylalanine-specific site of the enzyme, and the other type, to the tRNA(Phe)-specific binding site. They resemble alternative modes of a destabilized enzyme-product complex and are predicted on the basis of thermodynamic considerations. The two modes of binding of acyl-tRNA compete with each other. The attachment of Phe-tRNA(Phe) to the phenylalanine-specific site dominates. At equilibrium, this complex is present at a fourfold higher concentration than the other type of complex. The HNO2 deaminated Phe-tRNA(Phe) binds exclusively to the site specific for L-phenylalanine. On the contrary, Ile-tRNA(Phe) adds at 94.1% to the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) with this site leads to enzymatic hydrolysis into L-phenylalanine and tRNA(Phe). The complex involving the phenylalanine-specific site is hydrolytically unproductive. L-Phenylalanine acts as an activator of the hydrolysis by occupying the amino acid specific site and by shifting the equilibrium between the complexes toward the binding ot Phe-tRNA(Phe) at the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) at the phenylalanine-specific site does not interfere sterically with the binding of free tRNA(Phe). The sequential addition of free and aminoacylated tRNA(Phe) exhibits negative cooperativity. Such a mechanism could help to expel the product from the enzyme.
Amino Acyl-tRNA Synthetases↗
Importance of the anticodon sequence in the aminoacylation of tRNAs by methionyl-tRNA synthetase and by valyl-tRNA synthetase in an Archaebacterium.
The mode of recognition of tRNAs by aminoacyl-tRNA synthetases and translation factors is largely unknown in archaebacteria. To study this process, we have cloned the wild type initiator tRNA gene from the moderate halophilic archaebacterium Haloferax volcanii and mutants derived from it into a plasmid capable of expressing the tRNA in these cells. Analysis of tRNAs in vivo show that the initiator tRNA is aminoacylated but is not formylated in H. volcanii. This result provides direct support for the notion that protein synthesis in archaebacteria is initiated with methionine and not with formylmethionine. We have analyzed the effect of two different mutations (CAU-->CUA and CAU-->GAC) in the anticodon sequence of the initiator tRNA on its recognition by the aminoacyl-tRNA synthetases in vivo. The CAU-->CUA mutant was not aminoacylated to any significant extent in vivo, suggesting the importance of the anticodon in aminoacylation of tRNA by methionyl-tRNA synthetase. This mutant initiator tRNA can, however, be aminoacylated in vitro by the Escherichia coli glutaminyl-tRNA synthetase, suggesting that the lack of aminoacylation is due to the absence in H. volcanii of a synthetase, which recognizes the mutant tRNA. Archaebacteria lack glutaminyl-tRNA synthetase and utilize a two-step pathway involving glutamyl-tRNA synthetase and glutamine amidotransferase to generate glutaminyl-tRNA. The lack of aminoacylation of the mutant tRNA indicates that this mutant tRNA is not a substrate for the H. volcanii glutamyl-tRNA synthetase. The CAU-->GAC anticodon mutant is most likely aminoacylated with valine in vivo. Thus, the anticodon plays an important role in the recognition of tRNA by at least two of the halobacterial aminoacyl-tRNA synthetases.
Acylation↗
A single amidotransferase forms asparaginyl-tRNA and glutaminyl-tRNA in Chlamydia trachomatis.
Aminoacyl-tRNA is generally formed by aminoacyl-tRNA synthetases, a family of 20 enzymes essential for accurate protein synthesis. However, most bacteria generate one of the two amide aminoacyl-tRNAs, Asn-tRNA or Gln-tRNA, by transamidation of mischarged Asp-tRNA(Asn) or Glu-tRNA(Gln) catalyzed by a heterotrimeric amidotransferase (encoded by the gatA, gatB, and gatC genes). The Chlamydia trachomatis genome sequence reveals genes for 18 synthetases, whereas those for asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase are absent. Yet the genome harbors three gat genes in an operon-like arrangement (gatCAB). We reasoned that Chlamydia uses the gatCAB-encoded amidotransferase to generate both Asn-tRNA and Gln-tRNA. C. trachomatis aspartyl-tRNA synthetase and glutamyl-tRNA synthetase were shown to be non-discriminating synthetases that form the misacylated tRNA(Asn) and tRNA(Gln) species. A preparation of pure heterotrimeric recombinant C. trachomatis amidotransferase converted Asp-tRNA(Asn) and Glu-tRNA(Gln) into Asn-tRNA and Gln-tRNA, respectively. The enzyme used glutamine, asparagine, or ammonia as amide donors in the presence of either ATP or GTP. These results suggest that C. trachomatis employs the dual specificity gatCAB-encoded amidotransferase and 18 aminoacyl-tRNA synthetases to create the complete set of 20 aminoacyl-tRNAs.
Amino Acyl-tRNA Synthetases↗
Misacylation and editing by Escherichia coli valyl-tRNA synthetase: evidence for two tRNA binding sites.
Valyl-tRNA synthetase (ValRS) has difficulty discriminating between its cognate amino acid, valine, and structurally similar amino acids. To minimize translational errors, the enzyme catalyzes a tRNA-dependent editing reaction that prevents accumulation of misacylated tRNA(Val). Editing occurs with threonine, alanine, serine, and cysteine, as well as with several nonprotein amino acids. The 3'-end of tRNA plays a vital role in promoting the tRNA-dependent editing reaction. Valine tRNA having the universally conserved 3'-terminal adenosine replaced by any other nucleoside does not stimulate the editing activity of ValRS. As a result 3'-end tRNA(Val) mutants, particularly those with 3'-terminal pyrimidines, are stably misacylated with threonine, alanine, serine, and cysteine. Valyl-tRNA synthetase is unable to hydrolytically deacylate misacylated tRNA(Val) terminating in 3'-pyrimidines but does deacylate mischarged tRNA(Val) terminating in adenosine or guanosine. Evidently, a purine at position 76 of tRNA(Val) is essential for translational editing by ValRS. We also observe misacylation of wild-type and 3'-end mutants of tRNA(Val) with isoleucine. Valyl-tRNA synthetase does not edit wild-type tRNA(Val)(A76) mischarged with isoleucine, presumably because isoleucine is only poorly accommodated at the editing site of the enzyme. Misacylated mutant tRNAs as well as 3'-end-truncated tRNA(Val) are mixed noncompetitive inhibitors of the aminoacylation reaction, suggesting that ValRS, a monomeric enzyme, may bind more than one tRNA(Val) molecule. Gel-mobility-shift experiments to characterize the interaction of tRNA(Val) with the enzyme provide evidence for two tRNA binding sites on ValRS.
3' Untranslated Regions↗
Three modified nucleosides present in the anticodon stem and loop influence the in vivo aa-tRNA selection in a tRNA-dependent manner.
In Salmonella typhimurium seven tRNA species specific for leucine, proline and arginine have 1-methylguanosine (m1G) next to and 3' of the anticodon (position 37 of tRNA), five tRNA species specific for phenylalanine, serine, tyrosine, cysteine and tryptophan have 2-methylthio-N-6-(cis-hydroxy)isopentenyladenosine (ms2io6A) in the same position of the tRNA, and four tRNA species, specific for leucine and proline, have pseudouridine (Psi) as the last 3' nucleotide in the anticodon loop (position 38) or in the anticodon stem (positions 39 and 40). Mutants deficient in the synthesis of these modified nucleosides have been used to study their role in the first step of translation elongation, i.e. the aa-tRNA selection step in which the ternary complex (EF-Tu-GTP-aa-tRNA) binds at the cognate codon in the A-site on the mRNA programmed ribosome. We have found that the Psi present in the anticodon loop (position 38) stimulates the selection of tRNA specific for leucine whereas Psi in the anticodon stem did not affect the selection of tRNA specific for proline. The m1G37 strongly stimulates the rate of selection of the three tRNA species specific for proline and one tRNA species specific for arginine but has only minor or no effect on the selection of the three tRNA species specific for leucine. Likewise, the ms2io6A, present in the same position as m1G37 but in another subset of tRNA species, stimulates the selection of tRNA specific for tyrosine, stimulates to some extent also tRNA species specific for cysteine and tryptophan, but has no influence on the rate of selection of tRNA specific for phenylalanine. We conclude that function of m1G and ms2io6A present next to and 3' of the anticodon influences the in vivo aa-tRNA selection in a tRNA-dependent manner.
Anticodon↗
Nucleotides of the tRNA D-stem that play an important role in nuclear-tRNA export in Saccharomyces cerevisiae.
Nuclear export of tRNA in Saccharomyces cerevisiae involves Los1p and Arc1p. Los1p facilitates tRNA translocation across the nuclear pore complex whereas Arc1p plays a role in delivering some species of tRNA exiting the nucleus to their cognate aminoacyl-tRNA synthetases. Here, we show that mutations of C11 and G24 of the D-stem of the yeast tyrosine amber-suppressor tRNA have different effects on nuclear export of the tRNA. Changing G24 had no effect on export of the tRNA to the cytoplasm. In contrast, mutating C11 resulted in nuclear retention of the tRNA. Nuclear retention of the tRNA mutants was not due to lack of processing, since only the mature forms of the tRNA mutants were found. The fact that mutations of G24 did not affect export of the tRNA also indicates that the effect of mutating C11 is not due to gross alteration of the tertiary structure resulting from disruption of the C11/G24 base pair. Expression of Los1p and the mammalian tRNA export receptor exportin-t rescued nuclear export of the tRNA with changes at position 11. The export-defective mutations of the tRNA mutants were suppressed by introducing the complementary nucleotides at position 24. Taken together, these findings suggest that C11 is important for binding of the tRNA to the export receptor, and that this binding is influenced by the conformation of the base. Finally, the export-defective tRNA mutants described can be used as reporters to identify eukaryotic proteins involved in the nuclear-tRNA export process, and characterize the molecular interactions between known receptors and the tRNA substrate.
Base Sequence↗
The site of hydrolysis by rabbit reticulocyte peptidyl-tRNA hydrolase is the 3'-AMP terminus of susceptible tRNA substrates.
The preceding paper (Gross, M., Starn, T.K., Rundquist, C., Crow, P., White, J., Olin, A., and Wagner, T. (1992) J. Biol. Chem. 267, 2073-2079) reported the purification and partial characterization of rabbit reticulocyte peptidyl-tRNA hydrolase. In this article we demonstrate that, unlike bacterial and yeast peptidyl-tRNA hydrolase which act by deacylation, the reticulocyte enzyme hydrolyzes N-acylaminoacyl-tRNA to N-acylaminoacyl-AMP. Reticulocyte lysate has a separate enzyme, that we have isolated and termed aminoacyl-AMP deacylase, which hydrolyzes N-acylaminoacyl-AMP and aminoacyl-AMP, recycling the amino acid and nucleotide components. The action of this enzyme is relatively specific for the N-acylaminoacyl-AMP generated by peptidyl-tRNA hydrolase, since it is much less active with N-acylaminoacyl-adenosine and inactive with N-acylaminoacyl-ACCAC, N-acylaminoacyl-tRNA, or aminoacyl-tRNA. The tRNA product of peptidyl-tRNA hydrolase action is tRNA missing only its 3'-AMP terminus (tRNA(c-c)), since reaminoacylation requires tRNA nucleotidyltransferase but not CTP. The 3' exonucleolytic action of reticulocyte peptidyl-tRNA hydrolase is specific to susceptible tRNA substrates, since it does not hydrolyze CACCA, CACCA-N-acylamino acid, polyuridylic acid, or the 3' polyadenylate tail of globin mRNA, and, since its ability to hydrolyze Escherichia coli f[3H]Met-tRNA(fMet) is not reduced by excess 5 S or 28 S ribosomal RNA and is reduced only slightly by excess tRNA(c-c). Reticulocyte peptidyl-tRNA hydrolase also hydrolyzes th 3'-AMP terminus of deacylated tRNA. This property may explain why the 3'-terminal AMP of tRNA undergoes turnover in reticulocytes and reticulocyte lysate, since we find that such turnover in gel-filtered reticulocyte lysate is increased under conditions where aminoacylation is reduced.
Adenosine Monophosphate↗