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1.
Science ; 246(4937): 1595-7, 1989 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-2688091

RESUMO

The contribution of the anticodon to the discrimination between cognate and noncognate tRNAs by Escherichia coli Arg-tRNA synthetase has been investigated by in vitro synthesis and aminoacylation of elongator methionine tRNA (tRNA(mMet) mutants. Substitution of the Arg anticodon CCG for the Met anticodon CAU leads to a dramatic increase in Arg acceptance by tRNA(mMet). A nucleotide (A20) previously identified by others in the dihydrouridine loop of tRNA(Arg)s makes a smaller contribution to the conversion of tRNA(mMet) identity from Met to Arg. The combined anticodon and dihydrouridine loop mutations yield a tRNA(mMet) derivative that is aminoacylated with near-normal kinetics by the Arg-tRNA synthetase.


Assuntos
Anticódon/genética , RNA de Transferência Aminoácido-Específico/genética , RNA de Transferência de Arginina/genética , RNA de Transferência/genética , Arginina-tRNA Ligase/metabolismo , Sequência de Bases , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Metionina tRNA Ligase/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Especificidade por Substrato , Fagos T/genética , Transcrição Gênica
2.
Science ; 242(4879): 765-8, 1988 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-3055296

RESUMO

The anticodon has previously been shown to play a role in recognition of certain transfer RNAs by aminoacyl-tRNA synthetases; however, the extent to which this sequence dictates tRNA identity is generally unknown. To investigate the contribution of the anticodon to the identity of Escherichia coli methionine and valine tRNAs, in vitro transcripts of these tRNAs were prepared that contained normal and interchanged anticodon sequences. Transcripts containing wild-type tRNA sequences were excellent substrates for their respective cognate aminoacyl-tRNA synthetases and were effectively discriminated against by a variety of noncognate enzymes. The mutant tRNAs produced by switching the anticodon sequences lost their original tRNA identity and assumed an identity corresponding to the acquired anticodon sequence. These results indicate that the anticodon contains sufficient information to distinguish methionine and valine tRNAs with high fidelity.


Assuntos
Anticódon , RNA de Transferência Aminoácido-Específico/fisiologia , RNA de Transferência de Metionina/fisiologia , RNA de Transferência de Valina/fisiologia , RNA de Transferência , Aminoacilação de RNA de Transferência , Escherichia coli , Cinética , Metionina tRNA Ligase/metabolismo , Especificidade por Substrato , Valina-tRNA Ligase/metabolismo
8.
Proc Natl Acad Sci U S A ; 69(12): 3594-7, 1972 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-4566450

RESUMO

The structural requirements of E. coli formylmethionine tRNA for aminoacylation have been examined by chemical modification of the tRNA, followed by separation of the modified molecules into active and inactive components. Photooxidation of tRNA(fMet) at 50 degrees in the presence of methylene blue results in modification of two guanosine (G) residues in the acceptor stem, at positions no. 2 and no. 71 from the 5'-phosphate terminus. Both of these modifications are present in inactive molecules, but only the G residue at position no. 2 is modified in the acceptor stem of active molecules. Loss of methionine acceptance occurs with first-order kinetics, indicating that inactivation by modification of G residue no. 71 is independent of any other modifications taking place under these conditions. The presence of a modified G residue at position no. 2 in the acceptor stem of active photooxidized molecules shows that disruption of normal base-pairing in this region is not sufficient to inactivate tRNA(fMet). These data indicate that the inactivating modification at position no. 71 is lethal due to a specific alteration in the nucleotide base, rather than simply as a result of breaking a hydrogen-bonded base pair in the acceptor stem.


Assuntos
Escherichia coli/metabolismo , Metionina/metabolismo , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Acilação , Sequência de Bases , Cromatografia DEAE-Celulose , Formiatos/metabolismo , Oligonucleotídeos/isolamento & purificação , Fotoquímica , Ribonucleases/metabolismo , Temperatura
9.
Proc Natl Acad Sci U S A ; 66(2): 507-14, 1970 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-4917443

RESUMO

E. coli formylmethionyl tRNA (tRNA(fMet)) has been irradiated with ultraviolet light in the presence of Mg(2+) to the extent of 50 per cent inactivation of amino acid acceptance. Separation of active and inactive molecules after irradiation has shown that ultraviolet light modification of the uridine in the anticodon, the uridine in the small loop, the 4-thiouridine, and the pyrimidines in the double-stranded stem adjacent to the dihydrouridine loop has no effect on aminoacylation or transformylation. The ultraviolet light-induced inactivation of methionine acceptance by tRNA(fMet) is due almost entirely to modification of the cytidine residues in the 3'-terminal CCA-OH sequence.


Assuntos
Aminoácidos/metabolismo , Escherichia coli , Ligases/metabolismo , RNA de Transferência/farmacologia , Raios Ultravioleta , Isótopos de Carbono , Cromatografia DEAE-Celulose , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Formiatos/análise , Homocisteína/metabolismo , Leucovorina/metabolismo , Metionina/análise , Metionina/metabolismo , Nucleotídeos/análise , RNA de Transferência/isolamento & purificação , RNA de Transferência/efeitos da radiação , Efeitos da Radiação , Ribonucleases/farmacologia , Análise Espectral , Transferases/metabolismo
10.
Biochemistry ; 25(16): 4555-61, 1986 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-3094575

RESUMO

Four different structural regions of Escherichia coli tRNAfMet have been covalently coupled to E. coli methionyl-tRNA synthetase (MetRS) by using a tRNA derivative carrying a lysine-reactive cross-linker. We have previously shown that this cross-linking occurs at the tRNA binding site of the enzyme and involves reaction of only a small number of the potentially available lysine residues in the protein [Schulman, L. H., Valenzuela, D., & Pelka, H. (1981) Biochemistry 20, 6018-6023; Valenzuela, D., Leon, O., & Schulman, L. H. (1984) Biochem. Biophys. Res. Commun. 119, 677-684]. In this work, four of the cross-linked peptides have been identified. The tRNA-protein cross-linked complex was digested with trypsin, and the peptides attached to the tRNA were separated from the bulk of the tryptic peptides by anion-exchange chromatography. The tRNA-bound peptides were released by cleavage of the disulfide bond of the cross-linker and separated by reverse-phase high-pressure liquid chromatography, yielding five major peaks. Amino acid analysis indicated that four of these peaks contained single peptides. Sequence analysis showed that the peptides were cross-linked to tRNAfMet through lysine residues 402, 439, 465, and 640 in the primary sequence of MetRS. Binding of the tRNA therefore involves interactions with the carboxyl-terminal half of MetRS, while X-ray crystallographic data have shown the ATP binding site to be located in the N-terminal domain of the protein [Zelwer, C., Risler, J. L., & Brunie, S. (1982) J. Mol. Biol. 155, 63-81].(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Escherichia coli/enzimologia , Metionina tRNA Ligase/metabolismo , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência de Metionina , Sequência de Aminoácidos , Sítios de Ligação , Reagentes de Ligações Cruzadas/farmacologia , Lisina , Ligação Proteica , Succinimidas/farmacologia
11.
Biochemistry ; 25(15): 4450-6, 1986 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-3092857

RESUMO

The accessibility of nucleotides in Escherichia coli tRNAfMet to chemical and enzymatic probes in the presence and absence of methionyl-tRNA synthetase has been investigated. Dimethyl sulfate was used to probe the reactivity of cytosine and guanosine residues. The N-3 position of the wobble anticodon base, C34, was strongly protected from methylation in the tRNA-synthetase complex. A synthetase-induced conformational change in the anticodon loop was suggested by the enhanced reactivity of C32 in the presence of enzyme. Cytosine residues in the dihydrouridine loop and in the 3'-terminal CCA sequence showed little or no change in reactivity. Methylation of the N-7 position of guanosine residues G42, G52, and G70 was partially inhibited by the synthetase. Nuclease digestion of tRNAfMet with alpha-sarcin in the presence of 1-2 mM Mg2+ resulted in cleavage mainly at C71 in the acceptor stem and was strongly inhibited by synthetase. Other nuclease digestion experiments using the single strand specific nucleases RNase A and RNase T1 revealed weak protection of nucleotides in the D loop and strong protection of nucleotides in the anticodon on complex formation. The present data, together with previous structure-function studies on this system, indicate strong binding of methionyl-tRNA synthetase to the anticodon of tRNAfMet, leading to a change in the conformation of the anticodon loop and stem. We propose that this, in turn, produces more distant, and possibly relatively subtle, conformational changes in other parts of the tRNA structure that ultimately lead to proper orientation of the 3' terminus of the tRNA with respect to the active site of the enzyme.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Escherichia coli/enzimologia , Metionina tRNA Ligase/metabolismo , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência de Metionina , Sequência de Bases , Citidina , Guanosina , Cinética , Conformação de Ácido Nucleico , Radioisótopos de Fósforo , Ligação Proteica , Ribonuclease T1 , Ribonuclease Pancreático
12.
Nucleic Acids Res ; 18(2): 285-9, 1990 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-2109304

RESUMO

Recent evidence indicates that the anticodon may often play a crucial role in selection of tRNAs by aminoacyl-tRNA synthetases. In order to quantitate the contribution of the anticodon to discrimination between cognate and noncognate tRNAs by E. coli threonyl-tRNA synthetase, derivatives of the E. coli elongator methionine tRNA (tRNA(mMet)) containing wild type and threonine anticodons have been synthesized in vitro and assayed for threonine acceptor activity. Substitution of the threonine anticodon GGU for the methionine anticodon CAU increased the threonine acceptor activity of tRNA(mMet) by four orders of magnitude while reducing methionine acceptor activity by an even greater amount. These results indicate that the anticodon is the major element which determines the identity of both threonine and methionine tRNAs.


Assuntos
Anticódon/genética , Escherichia coli/genética , Genes Bacterianos , Genes Sintéticos , Metionina , Aminoacil-RNA de Transferência/genética , RNA de Transferência/genética , Treonina , Sequência de Bases , Escherichia coli/enzimologia , Cinética , Metionina tRNA Ligase/isolamento & purificação , Metionina tRNA Ligase/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos , Treonina-tRNA Ligase/isolamento & purificação , Treonina-tRNA Ligase/metabolismo , Transcrição Gênica
13.
Fed Proc ; 43(15): 2977-80, 1984 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-6389181

RESUMO

In previous work we identified several specific sites in Escherichia coli tRNAfMet that are essential for recognition of this tRNA by E. coli methionyl-tRNA synthetase (MetRS) (EC 6.1.1.10). Particularly strong evidence indicated a role for the nucleotide base at the wobble position of the anticodon in the discrimination process. We have now investigated the aminoacylation activity of a series of tRNAfMet derivatives containing single base changes in each position of the anticodon. In addition, derivatives containing permuted sequences and larger and smaller anticodon loops have been prepared. The variant tRNAs have been enzymatically synthesized in vitro by using T4 RNA ligase (EC 6.5.1.3). Base substitutions in the wobble position have been found to reduce aminoacylation rates by at least five orders of magnitude. Derivatives having base substitutions in the other two positions of the anticodon are aminoacylated 55-18,500 times slower than normal. Nucleotides that have specific functional groups in common with the normal anticodon bases are better tolerated at each of these positions than those that do not. A tRNAfMet variant having a six-membered loop containing only the CA sequence of the anticodon is aminoacylated still more slowly, and a derivative containing a five-membered loop is not measurably active. The normal loop size can be increased by one nucleotide with a relatively small effect on the rate of aminoacylation, which indicates that the spatial arrangement of the nucleotides is less critical than their chemical nature. We conclude from these data that recognition of tRNAfMet requires highly specific interactions of MetRS with functional groups on the nucleotide bases of the anticodon sequence. Several other aminoacyl-tRNA synthetases are known to require one or more anticodon bases for efficient aminoacylation of their tRNA substrates, and data from other laboratories suggest that anticodon sequences may be important for accurate discrimination between cognate and noncoagnate tRNAs by these enzymes.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Metionina tRNA Ligase/metabolismo , RNA de Transferência/metabolismo , Anticódon , Sequência de Bases , Escherichia coli/enzimologia , Cinética , Conformação de Ácido Nucleico , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
14.
Biochemistry ; 24(25): 7309-14, 1985 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-3910101

RESUMO

A derivative of Escherichia coli tRNAfMet containing an altered anticodon sequence, CUA, has been enzymatically synthesized in vitro. The variant tRNA was prepared by excision of the normal anticodon, CAU, in a limited digestion of intact tRNAfMet with RNase A, followed by insertion of the CUA sequence into the anticodon loop with T4 RNA ligase and polynucleotide kinase. The altered methionine tRNA showed a large enhancement in the rate of aminoacylation by glutaminyl-tRNA synthetase and a large decrease in the rate of aminoacylation by methionyl-tRNA synthetase. Measurement of kinetic parameters for the charging reaction by the cognate and noncognate enzymes revealed that the modified tRNA is a better acceptor for glutamine than for methionine. The rate of mischarging is similar to that previously reported for a tryptophan amber suppressor tRNA containing the anticodon CUA, su+7 tRNATrp, which is aminoacylated with glutamine both in vivo and in vitro [Yaniv, M., Folk, W. R., Berg, P., & Soll, L. (1974) J. Mol. Biol. 86, 245-260; Yarus, M., Knowlton, R. E., & Soll, L. (1977) in Nucleic Acid-Protein Recognition (Vogel, H., Ed.) pp 391-408, Academic Press, New York]. The present results provide additional evidence that the specificity of aminoacylation by glutaminyl-tRNA synthetase is sensitive to small changes in the nucleotide sequence of noncognate tRNAs and that uridine in the middle position of the anticodon is involved in the recognition of tRNA substrates by this enzyme.


Assuntos
Escherichia coli/genética , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência de Metionina , Trifosfato de Adenosina/metabolismo , Anticódon , Sequência de Bases , Radioisótopos de Carbono , Glutamina/metabolismo , Indicadores e Reagentes , Cinética , Conformação de Ácido Nucleico , Oligorribonucleotídeos/síntese química , Radioisótopos de Fósforo , Aminoacil-RNA de Transferência/síntese química
15.
Biochemistry ; 15(26): 5769-75, 1976 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-827308

RESUMO

Chemical modification of Escherichia coli tRNAfMet with 1 M chloroacetaldehyde, pH 5.5-6.0 at 25 degrees C, has been found to result in alteration of six cytidine and five adenosine residues in the molecule. The modified cytidine residues are the same as those previously found to be reactive with sodium bisulfite at pH 6.0. The accessible adenosine residues are A36 in the anticodon, A58 in the T psi C loop, and A73, A74, and A77 in the 3; terminal sequence. No modification of adenosine residues in the dihydrouridine or variable loops or of adenosine residues on the 3' side of the anticodon loop could be detected. Treatment of fMet-tRNAfMet with chloracetaldehyde gave the same pattern of midofication as was observed with deacylated tRNAfMet. Chemical modification of E. coli tRNAfMet with 2 sodium bisulfite, pH 7.0 at 25 degrees C, resulted in selective modification of exposed uridine residues in the tRNA. Only three sites were found to be reactive: U18 in the dihydrouridine loop, U37 in the anticodon, and U48 in the variable loop. The overall pattern of chemical modification of tRNAfMet is very similar to that found by others for yeast tRNAPhe, supporting the idea that many of the tertiary interactions in the two tRNAs are the same. The adenosine residue at position 58 in the center of the T psi C loop of the initiator tRNA shows unusual reactivity, however, being modified by chloroacetaldehyde at the same rate as the 3' terminal adenosine residue. This result is in sharp contrast to the uniform resistance of nucleotides in the T psi C loop of yeast tRNAPhe to chemical modification.


Assuntos
Escherichia coli/metabolismo , RNA de Transferência , Acetaldeído/análogos & derivados , Sítios de Ligação , Cinética , N-Formilmetionina , Conformação de Ácido Nucleico , Oligorribonucleotídeos/análise , Pâncreas/enzimologia , RNA de Transferência/metabolismo , Ribonuclease T1 , Ribonucleases
16.
J Biol Chem ; 252(3): 814-9, 1977 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-14133

RESUMO

Treatment of Escherichia coli formylmethionine tRNA with 2 M sodium bisulfite, pH 7.0, in 10 mM MgCl2 at 25 degrees results in formation of uridine/bisulfite adducts at U18 in the dihydrouridine loop, U37 in the anticodon, and U48 in the variable loop. Two products, corresponding to the two diastereoisomers of 5,6-dihydrouridine-6-sulfonate, are formed at each reactive site in the tRNA. Although none of the modifications cause complete loss of methionine acceptor activity, the modified tRNA is amino-acylated at a reduced rate and has a decreased affinity for E. coli methionyl-tRNA synthetase. Aminoacylation of [35S]bisulfite-labeled tRNAfMet with a limiting amount of purified enzyme followed by separation of the acylated and unacylated molecules and structural analysis has shown that the presence of a specific diastereoisomer of the uridine/bisulfite adduct in the anticodon base U37 alters the kinetic parameters for aminoacylation of tRNAfMet.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Anticódon/metabolismo , Escherichia coli/metabolismo , Metionina tRNA Ligase/metabolismo , RNA de Transferência/metabolismo , Sequência de Bases , Sítios de Ligação , Concentração de Íons de Hidrogênio , Cinética , N-Formilmetionina , Sulfitos
17.
Proc Natl Acad Sci U S A ; 88(9): 3872-6, 1991 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-2023934

RESUMO

An assay based on the initiation of protein synthesis in Escherichia coli has been used to explore the role of the anticodon in tRNA identity in vivo. Mutations were introduced into the initiator tRNA to change the wild-type anticodon from CAU (methionine) to GAU (isoleucine), GAC (valine), and GAA (phenylalanine), where each derivative differs from the preceding by a single base change in the anticodon (underlined). These changes were sufficient to cause the mutant tRNAs to be aminoacylated by the corresponding aminoacyl-tRNA synthetases based on the amino acid inserted into protein from complementary initiation codons. Construction of additional single base anticodon variants (GUU, GGU, GCC, GUC, GCA, and UAA) showed that all three anticodon bases specify isoleucine and phenylalanine identity and that both the middle and the third anticodon bases are important for valine identity in vivo.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Anticódon , Isoleucina/metabolismo , Iniciação Traducional da Cadeia Peptídica , Fenilalanina/metabolismo , RNA de Transferência de Metionina/metabolismo , Valina/metabolismo , Sequência de Bases , Escherichia coli/genética , Técnicas In Vitro , Isoleucina-tRNA Ligase/metabolismo , Dados de Sequência Molecular , Fenilalanina-tRNA Ligase/metabolismo , Especificidade por Substrato , Valina-tRNA Ligase/metabolismo
18.
J Biol Chem ; 250(2): 542-7, 1975 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-1089645

RESUMO

Escherichia coli formylmethionly-tRNA-tMet is unique among N-acylaminoacyl-tRNAs in its resistance to cleavage by peptidyl-tRNA hydrolase. Chemical modification of tRNA-fMet with sodium bisulfite converts fMet-tRNA-fMet into a good substrate for the hydrolase. The products of the enzymatic cleavage are free tRNA-fMet and formylmethionine. Bisulfite treatment produces cytidine to uridine base changes at several sites in the tRNA structure. One of these modifications results in formation of a new hydrogen-bonded base pair at the end of the acceptor stem of tRNA-fMet. We have shown that this modification is responsible for the observed change in biological activity. Enzymatic cleavage appears to be facilitated by the presence of a 5-terminal phosphate at the end of a fully base-paired acceptor stem, because removal of the 5-phosphate group from N-acetylphenylalanyl-tRNA-Phe or bisulfite-modified fMet-tRNA-FMet reduced the rate of hydrolysis of these substrates. The unpaired base at the 5 terminus of unmodified fMet-tRNA-fMet appears to reduce susceptibility of the tRNA to hydrolytic attack both by positioning the 5-phosphate in an unfavorable orientation and by directly interfering with enzymatic binding. The unusual structure of the acceptor stem of this E. coli tRNA thus plays a critical role in maintaining the viability of the organism by preventing enzymatic cleavage of the fMet group from the bacterial initiator tRNA.


Assuntos
Escherichia coli/enzimologia , Hidrolases , RNA Bacteriano , RNA de Transferência , Aminoacil-tRNA Sintetases , Sequência de Bases , Sítios de Ligação , Cromatografia , Hidrolases/isolamento & purificação , Metionina/análogos & derivados , Conformação de Ácido Nucleico , Peptídeos , Fenilalanina/análogos & derivados , Sulfitos
19.
Proc Natl Acad Sci U S A ; 80(22): 6755-9, 1983 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-6359155

RESUMO

Previous work from our laboratory identified several specific sites in Escherichia coli tRNAfMet that are essential for recognition of this tRNA by E. coli methionyl-tRNA synthetase (EC 6.1.1.10). Particularly strong evidence indicated a role for the nucleotide base at the wobble position of the anticodon in the discrimination process. To further investigate the structural requirements for recognition in this region, we have synthesized a series of tRNAfMet derivatives containing single base changes in each position of the anticodon. In addition, derivatives containing permuted sequences and larger and smaller anticodon loops have been prepared. The variant tRNAs have been enzymatically synthesized in vitro. The procedure involves excision of the normal anticodon, CAU, by limited digestion of intact tRNAfMet with pancreatic RNase. This step also removes two nucleotides from the 3' CpCpA end. T4 RNA ligase is used to join oligonucleotides of defined length and sequence to the 5' half-molecule and subsequently to link the 3' and modified 5' fragment to regenerate the anticodon loop. The final step of the synthesis involves repair of the 3' terminus with tRNA nucleotidyltransferase. The synthetic derivative containing the anticodon CAU is aminoacylated with the same kinetics as intact tRNAfMet. Base substitutions in the wobble position reduce aminoacylation rates by at least five orders of magnitude. The rates of aminoacylation of derivatives having base substitutions in the other two positions of the anticodon are 1/55 to 1/18,500 times normal. Nucleotides that have specific functional groups in common with the normal anticodon bases are better tolerated at each of these positions than those that do not. A tRNAfMet variant having a six-membered loop containing only the CA sequence of the anticodon is aminoacylated still more slowly, and a derivative containing a five-membered loop is not measurably active. The normal loop size can be increased by one nucleotide with a relatively small effect on the rate of aminoacylation, indicating that the spatial arrangement of the nucleotides is less critical than their chemical nature. We conclude from these data that recognition of tRNAfMet requires highly specific interactions of methionyl-tRNA synthetase with functional groups on the nucleotide bases of the anticodon sequence.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Anticódon , Escherichia coli/genética , Metionina tRNA Ligase/metabolismo , RNA de Transferência , Sequência de Bases , Escherichia coli/enzimologia , Cinética
20.
Biochemistry ; 26(22): 7113-21, 1987 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-3122828

RESUMO

A new method has been developed to couple a lysine-reactive cross-linker to the 4-thiouridine residue at position 8 in the primary structure of the Escherichia coli initiator methionine tRNA (tRNAfMet). Incubation of the affinity-labeling tRNAfMet derivative with E. coli methionyl-tRNA synthetase (MetRS) yielded a covalent complex of the protein and nucleic acid and resulted in loss of amino acid acceptor activity of the enzyme. A stoichiometric relationship (1:1) was observed between the amount of cross-linked tRNA and the amount of enzyme inactivated. Cross-linking was effectively inhibited by unmodified tRNAfMet, but not by noncognate tRNAPhe. The covalent complex was digested with trypsin, and the resulting tRNA-bound peptides were purified from excess free peptides by anion-exchange chromatography. The tRNA was then degraded with T1 ribonuclease, and the peptides bound to the 4-thiouridine-containing dinucleotide were purified by high-pressure liquid chromatography. Two major peptide products were isolated plus several minor peptides. N-Terminal sequencing of the peptides obtained in highest yield revealed that the 4-thiouridine was cross-linked to lysine residues 402 and 439 in the primary sequence of MetRS. Since many prokaryotic tRNAs contain 4-thiouridine, the procedures described here should prove useful for identification of peptide sequences near this modified base when a variety of tRNAs are bound to specific proteins.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Aminobenzoatos/síntese química , Reagentes de Ligações Cruzadas/síntese química , Escherichia coli/enzimologia , Lisina , Metionina tRNA Ligase/metabolismo , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência de Metionina , Succinimidas/síntese química , Tiouridina , Aminobenzoatos/metabolismo , Sequência de Bases , Indicadores e Reagentes , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Succinimidas/metabolismo , para-Aminobenzoatos
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