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PubMed1982

The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules.

The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM). Kirromycin protects both EF-Tu.GDP and EF-Tu.GTP against modification with TPCK. Binding of aminoacyl-tRNA added at increasing concentrations to a solution of 40 microM EF-Tu.GDP.kirromycin complex re-exposes the TPCK target site on the protein. However, when the aminoacyl-tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl-tRNA. By contrast, addition of uncharged tRNA or N- acetylaminoacyl -tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF-Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl-tRNA, and N- acetylaminoacyl -tRNA. Support for this assumption is provided by measuring the modification of EF-Tu.GDP with the sulfhydryl reagent NEM. Moreover, NEM modification also indicates an additional tRNA binding site on EF-Tu.GTP.kirromycin, which could not be detected with TPCK. Mapping of the tryptic peptides of EF-Tu.GDP labeled with [14C]TPCK revealed only one target site for this agent, i.e., cysteine-81. Modification occurred at the same site in the presence and in the absence of kirromycin and uncharged tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Bacterial Agents↗

PubMed1995

Nucleotides of tRNA (Glu) involved in recognition by barley chloroplast glutamyl-tRNA synthetase and glutamyl-tRNA reductase.

The biosynthesis of delta-aminolevulinate (ALA), via the C-5 pathway, requires tRNA(Glu) as a cofactor for the glutamyl tRNA(Glu) synthetase and the glutamyl tRNA(Glu) reductase which are the first two enzymes in this three step pathway. These two enzymes form a ternary complex with the tRNA(Glu) in Chlamydomonas reinhardtii suggesting that the recognition elements on the tRNA cofactor are different for each enzyme. Chemical modification and comparative studies with tRNA(Glu)s from a number of species were used to determine the nucleotides involved in the recognition of the barley chloroplast tRNA(Glu) by the barley enzymes. The barley chloroplast tRNA(Glu) is chemically modified both before and after ligation to glutamate with monobromobimane or CNBr. The chemically modified tRNA(Glu) is a poor substrate for the glutamyl-tRNA synthetase and the chemically modified glutamyl-tRNA(Glu) is used as a substrate for glutamyl-tRNA(Glu) reductase. The tRNA(Glu) from the chloroplasts if barley, Chlamydomonas reinhardtii, tobacco, cucumber, wheat and spinach and tRNA(Glu) from Synechocystis PCC6803, Escherichia coli, barley germ and bakers yeast and the barley chloroplast tRNA(Gln) are all effective substrates for the barley chloroplast glutamyl-tRNA synthetase. A comparison of the sequences of these tRNAs shows 19 conserved bases and five of these bases, G10, A26, U34, U35 and A37 are suggested as recognition elements of barley glutamyl tRNA(Glu) synthetase by assuming a similar binding orientation as in the crystal structure of the E. coli tRNA(Gln) GlnRS complex. The glutamyl-tRNA(Glu) from E. coli, bakers yeast and barley germ and the barley chloroplast glutamyl-tRNA(Gln) are not effective substrates for the barley chloroplast glutamyl-tRNA(Glu) reductase. A comparison of the sequences of these four tRNA species with the sequences of the tRNA(Glu) species that can be used as substrate by the glutamyl-tRNA(Glu) reductase yields seven common differences in the primary sequence. These 7 nucleotides, A7-U66, U29-A41, A53-U61, and U72 are expected to be required for recognition by the barley chloroplast glutamyl-tRNA(Glu) reductase.

Aldehyde Oxidoreductases↗

PubMed1984

An antisuppressor mutation of Schizosaccharomyces pombe affects the post-transcriptional modification of the "wobble" base in the anticodon of tRNAs.

The screening of antisuppressor mutants of the yeast Schizosaccharomyces pombe has been successfully accomplished with high resolution liquid chromatographic methods for the analysis of tRNA nucleosides. Antisuppressor mutations reduce or abolish the function of nonsense suppressor-tRNAs or other informational suppressors. Nonradioactive or 35S-labeled unfractionated tRNA from various strains was digested to nucleosides and analyzed by high performance liquid chromatography. The mutant sin3 has lost the nucleoside 5-(methoxycarbonylmethyl)-2-thiouridine from its tRNA in comparison to parental strains. In eukaryotes this nucleoside is found at the first position of the anticodon (wobble position) in several isoacceptor tRNAs that preferentially recognize codons ending with adenosine. The sin3 mutation reduces the efficiency of UGA and UAA suppressor tRNASer and suppressor tRNALeu. The genetic cosegregation of modification loss, antisuppressor phenotype, and a change in cell size is demonstrated. This indicates that a single mutation in the structural gene for a tRNA modification enzyme causes the three different phenotypes.

Anticodon↗

PubMed1976

Analysis of the transfer RNA population of mouse mammary glands infected with a latent mammary tumor virus.

Mammary gland transfer RNA's (tRNA'S) of CEH mice infected with mammary tumor virus were analyzed in the preneoplastic state and compared to tRNAs of virus-free C3Hf mice and another uninfected strain, C57BL/6, which is completely resistant to cancer. This quantitative study was based on the ability of each tRNA to fix its corresponding amino acid. The amount of each of the 17 tRNA's tested was identical for the three mammary glands. In addition, tRNA populations during lactation correlated with the amino acids incorporated into the lactoproteins synthesized, which indicates adapation of the tRNA's to protein biosynthesis. Qualitative chromatographic studies on reverse phase capillary columns Type 5 of 10 aminoacyl-tRNA's did not reveal any difference in the isoacceptor elution profiles. This shows that no new isoaccepting tRNA is associated with the mammary tumor virus at that stage, and that no viral modification of a host tRNA has occurred.

Amino Acids↗

PubMed1996

Rp-deoxy-phosphorothioate modification interference experiments identify 2'-OH groups in RNase P RNA that are crucial to tRNA binding.

Ribose 2'-hydroxyls make a key contribution to the enormous structural and functional potential of RNA molecules. Here, we report the identification of 2'-deoxy modifications in the catalytic RNA subunit of RNase P from Escherichia coli that interfere with tRNA binding. This was accomplished by modification interference employing pools of RNase P RNA that carried a low level of Rp-deoxy-phosphorothioate (Rp-deoxyNMPalpha(S) ) modifications randomly distributed over its 380 nt. A gel retardation assay allowed us to separate RNase P RNA pools into tRNA-binding and nonbinding fractions. Differences in the intensity of phosphorothioate-specific iodine hydrolysis patterns of the two RNA fractions revealed positions where the Rp-deoxyNMPalpha(S) modification interferes with tRNA binding. A comparison with interference patterns obtained for the Rp-NMPalpha(S) modification alone has identified some 20 positions in the backbone of E. coli RNase P RNA where the functional defect caused by the Rp-deoxyNMPalpha(S) double modification is attributable to the 2'-deoxy modification (or possibly the C5 methyl group in the case of U residues because we used deoxyTMPalpha(S) for partial substitution of UMP). Most of the corresponding 2'-OH functions were localized in regions that have been reported to crosslink to photoreactive tRNA derivatives, suggesting that these 2'-hydroxyls are located along the tRNA binding interface of E. coli RNase P RNA. Our results indicate that the modification interference approach applied here will be useful generally to identify structurally and functionally important 2'-hydroxyls in large RNAs and ribozymes.

Animals↗

PubMed1970

Modifications du renouvellement de l'amp final du tRNA dans le foie de rat sous l'influence de divers inhibiteurs de synthese.

The turnover of terminal AMP of rat liver tRNA relative to that of internal AMP was studied in presence of various inhibitors. Actinomycin D and aflatoxin B(1), which strongly depress transcription in liver, lead to an increase of the specific radioactivity of external AMP/specific radioactivity of internal AMP ratio. On the contrary, drugs which inhibit the in vivo incorporation of aminoacids determine a significant decrease of this ratio.

Journal Article↗

PubMed1975

Effect of sodium bisulfite modification on the arginine acceptance of E. coli tRNA Arg.

Escherichia coli tRNA Arg was treated with sodium bisulfite to convert exposed cytosine residues to uracil. This treatment resulted in the loss of amino acid acceptance of the tRNA Arg with pseudo first-order reaction kinetics. The active and inactive molecules were separated after about 60e active and inactive molecules were separated after about 60 percent inactivation and analyzed for U in various positions by finger-printing of the oligonucleotides produced by nucleases. The results show that C to U base transitions in the dihydrouridine loop and in the CCA terminus have no effect on the aminoacylation of this tRNA. Deamination of a cytosine residue at the second position of the anticodon resulted in the loss of amino acid acceptor activity of arginine transfer RNA.

Amino Acyl-tRNA Synthetases↗

PubMedDate not supplied

[Photoaffinity modification of Escherichia coli ribosomes near the tRNA-binding centers by tRNAPhe derivatives carrying arylazido groups on guanosine residues].

Photoreactive derivatives of tRNAPhe (E. coli) bearing arylazido groups scattered statistically over guanosine residues (azido-tRNA) were applied for affinity labelling of E. coli ribosomes in elongation factor-dependent or factor-free model systems mimicking different steps of elongation. It is shown that UV-irradiation of the corresponding complexes of ribosomes with tRNA derivatives results in labelling of both subunits, the 30S one is labelled preferentially. In all experiments only ribosomal proteins were labelled. Comparison of the sets of proteins labelled by tRNA derivatives in different states at P-site allowed us to draw important conclusions concerning the influence of peptidyl moiety and of the occupancy of the A-site with aminoacyl- or peptidyl-tRNA on the arrangement of tRNA located at the P-site. Three of the 30S proteins--S11, S13 S19--are labelled with tRNA derivatives located at P-site in all states.

Azides↗

PubMed2004

An aminoacyl-tRNA synthetase that specifically activates pyrrolysine.

Pyrrolysine, the 22nd cotranslationally inserted amino acid, was found in the Methanosarcina barkeri monomethylamine methyltransferase protein in a position that is encoded by an in-frame UAG stop codon in the mRNA. M. barkeri encodes a special amber suppressor tRNA (tRNA(Pyl)) that presumably recognizes this UAG codon. It was reported that Lys-tRNA(Pyl) can be formed by the aminoacyl-tRNA synthetase-like M. barkeri protein PylS [Srinivasan, G., James, C. M. & Krzycki, J. A. (2002) Science 296, 1459-1462], whereas a later article showed that Lys-tRNA(Pyl) is synthesized by the combined action of LysRS1 and LysRS2, the two different M. barkeri lysyl-tRNA synthetases. Pyrrolysyl-tRNA(Pyl) formation was presumed to result from subsequent modification of lysine attached to tRNA(Pyl). To investigate whether pyrrolysine can be directly attached to tRNA(Pyl) we chemically synthesized pyrrolysine. We show that PylS is a specialized aminoacyl-tRNA synthetase for charging pyrrolysine to tRNA(Pyl); lysine and tRNA(Lys) are not substrates of the enzyme. In view of the properties of PylS we propose to name this enzyme pyrrolysyl-tRNA synthetase. In contrast, the LysRS1:LysRS2 complex does not recognize pyrrolysine and charges tRNA(Pyl) with lysine. These in vitro data suggest that Methanosarcina cells have two pathways for acylating the suppressor tRNA(Pyl). This would ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon.

Adenosine Monophosphate↗

PubMed1995

Inhibition of HIV-1 in CEM cells by a potent TAR decoy.

TAR decoys are short RNA oligonucleotides, corresponding to the HIV TAR sequence, which inhibit HIV expression and replication by blocking the binding of the HIV regulatory protein Tat to the authentic TAR region. In previous studies, TAR decoys expressed from a tRNA polIII promoter were moderately effective at inhibiting HIV in isolated human T cell lines and less effective at inhibiting HIV in peripheral blood CD4+ T cells. In this study, a series of modifications was introduced into the tRNA expression cassette in order to improve their effectiveness. These modifications included the addition of sequences which are predicted to have stem-loop secondary structures and addition of a wild-type tRNA processing site. TAR decoy RNA expressed in CEM cells from modified tRNA-based expression cassettes yielded five- to 20-fold more TAR transcripts than unmodified tRNA-based expression cassettes. HIV replication, as measured by a flow cytometric method to quantify intracellular viral p24 expression, was significantly reduced in polyclonal populations of CEM cells expressing a modified tRNA-TAR transcript that contains a wild-type tRNA processing site and stem-loops 5' and 3' to the TAR sequence. Similar modifications to the tRNA expression cassette also increased the intracellular concentration of a random test oligonucleotide, indicating that this improved expression system may also be useful for antisense and ribozyme based gene inhibition strategies.

Antiviral Agents↗

PubMed2006

Impact of forced selection of tRNAs on HIV-1 replication and genome stability highlight preferences for selection of certain tRNAs.

Human immunodeficiency virus (HIV-1) exclusively selects tRNA(Lys,3) as the primer for initiation of reverse transcription. How and why HIV-1 selects the tRNA is unresolved. To address this issue, we have generated HIV-1 in which the PBS was changed to be complementary to alternative tRNAs. In this study, we report on HIV-1 that have the PBS mutated to be complementary to tRNA(Thr), tRNA(Phe), tRNA(Ser) and tRNA(Tyr). Virus with a PBS complementary to tRNA(Thr) grew slightly slower than the wild type virus and maintained the PBS for an extended culture period before finally reverting back to utilize tRNA(Lys,3). In contrast, viruses with a PBS complementary to tRNA(Phe) or tRNA(Ser) rapidly reverted to utilize tRNA(Lys,3) following limited in vitro replication, while a virus with a PBS complementary to tRNA(Tyr) had severely compromised infectivity and did not productively infect a continuous T cell line (SupT1) or human peripheral blood mononuclear cells (PBMC). Modification of the A-loop region to be complementary to tRNA(Thr) with the mutation in the PBS to be complementary to tRNA(Thr) resulted in a virus that could stably utilize this tRNA while the modification of the A-loop to be complementary to the anticodon of tRNA(Ser) did not allow the virus to stably utilize tRNA(Ser). Modification of the A-loop region to be complementary to the anticodon of tRNA(Phe) severely impacted the replication of this virus. Finally, the modification of the A-loop region to be complementary to tRNA(Tyr) did not rescue the virus with a PBS complementary to tRNA(Tyr). The results of these studies demonstrate the diverse effects that alteration of the PBS to force selection of alternative primers have on HIV-1 replication and provide a framework to understand the dynamics of primer selection.

Base Sequence↗

PubMed2002

Imbalance of tRNA(Pro) isoacceptors induces +1 frameshifting at near-cognate codons.

Increased expression of the CCU/CCA/CCG-decoding tRNA(Pr)(o)3 on a multicopy plasmid leads to suppression of several +1 frameshift mutations in Salmonella enterica serovar Typhimurium. Systematic analysis of the site of frameshifting indicates that excess tRNA(Pr)(o)3 promotes near-cognate decoding at CCC codons. Re-phasing of the reading frame can be achieved by a subsequent slippage of the tRNA onto a cognate codon in the +1 reading frame. Frameshifting appears to be due to an imbalance of CCC-cognate and near-cognate tRNAs, as the effect of excess tRNA(Pr)(o)3 on reading frame maintenance can be reversed by increasing simultaneously the concentration of the cognate tRNA(Pr)(o)2. Finally, the cmo5U modification present at position 34 of tRNA(Pr)(o)3, which allows this tRNA to decode CCU in addition to CCG and CCA, also affects frameshifting, indicating that the ability of the near-cognate tRNA to decode a cognate codon efficiently in the alternative reading frame is important for re-phasing of the reading frame.

Alcohol Oxidoreductases↗

PubMed2004

Gene expression in Chromobacterium violaceum.

The repertoire of 4,431 open reading frames (ORFs), eight rRNA operons and 98 tRNA genes of Chromobacterium violaceum must be expressed in a regulated manner for successful adaptation to a wide variety of environmental conditions. To accomplish this feat, the organism relies on protein machineries involved in transcription, RNA processing and translation. Analysis of the C. violaceum genome showed that transcription initiation, elongation and termination are performed by the five well-known RNA polymerase subunits, five categories of sigma 70 factors, one sigma 54 factor, as well as six auxiliary elongation and termination factors. RNA processing is performed by a variety of endonucleases and exonucleases, such as ribonuclease H, ribonuclease E, ribonuclease P, and ribonuclease III, in addition to poly(A) polymerase and specific methyltransferases and pseudouridine synthases. ORFs for all ribosomal proteins, except S22, were found. Only 19 aminoacyl-tRNA synthetases were found, in addition to three aminoacyl-tRNA synthetase-related proteins. Asparaginyl-tRNA (Asn) is probably obtained by enzymatic modification of a mischarged aminoacyl-tRNA. The translation factors IF-1, IF-2, IF-3, EF-Ts, EF-Tu, EF-G, RF-1, RF-2 and RF-3 are all present in the C. violaceum genome, although the absence of selB suggests that C. violaceum does not synthesize selenoproteins. The components of trans-translation, tmRNA and associated proteins, are present in the C. violaceum genome. Finally, a large number of ORFs related to regulation of gene expression were also found, which was expected, considering the apparent adaptability of this bacterium.

Adaptation, Physiological↗

PubMed2006

A heterochromatin barrier partitions the fission yeast centromere into discrete chromatin domains.

BACKGROUND: Centromeres are cis-acting chromosomal domains that direct kinetochore formation, enabling faithful chromosome segregation. Centromeric regions of higher eukaryotes are structurally complex, consisting of various epigenetically modified chromatin types including specialized chromatin at the kinetochore itself, pericentromeric heterochromatin, and flanking euchromatin. Although the features necessary for the establishment and maintenance of discrete chromatin domains remain poorly understood, two models have been proposed based either on the passive convergence of competing activities involved in individual domain formation or, alternatively, on the action of specific genomic sequences and associated proteins to actively block the propagation of one chromatin type into another. RESULTS: Functional analysis of centromeric sequences located at the intersection of Schizosaccharomyces pombe central core chromatin and outer repeat heterochromatin identified a chromatin barrier that contains a transfer RNA (tRNA) gene. Deletion or modification of the barrier sequences result in the propagation of pericentromeric heterochromatin beyond its normal boundary. The tRNA gene is transcriptionally active, and barrier activity requires sequences necessary for RNA polymerase III transcription. Moreover, absence of the barrier results in abnormal meiotic chromosome segregation. CONCLUSIONS: The identification of DNA sequences with chromatin barrier activity at the fission yeast centromere provides a model for establishment of centromeric chromatin domains in higher eukaryotes.

Base Sequence↗

PubMed1993

Chemical modification and mutagenesis studies on zinc binding of aminoacyl-tRNA synthetases.

Thermus thermophilus methionyl-tRNA synthetase consists of two identical subunits with a potential Zn(2+)-binding sequence of Cys-X2-Cys-X13-Cys-X2-His (Nureki, O., Muramatsu, T., Suzuki, K., Kohda, D., Matsuzawa, H., Ohta, T. Miyazawa, T., and Yokoyama, S. (1991) J. Biol. Chem. 266, 3268-3277). Upon chemical modification of the 3 Cys residues of T. thermophilus MetRS with sodium p-(hydroxymercuri)phenylsulfonate, one Zn2+ ion was released from one subunit of the molecule, as monitored with 4-(2-pyridylazo)resorcinol. Site-directed mutagenesis of Cys and His residues in the Zn(2+)-binding sequence reduced the aminoacylation activity; the kcat value was markedly decreased, and the Km values for L-methionine and tRNAf(Met) were increased. Similarly, Cys modification released two Zn2+ ions from T. thermophilus and Escherichia coli isoleucyl-tRNA synthetases and E. coli threonyl-tRNA synthetase, which have Zn(2+)-binding motifs, and impaired their activities. By contrast, three other aminoacyl-tRNA synthetases that lack Zn(2+)-binding motif neither released Zn2+ ion nor lost their activities upon Cys modification. These results indicate that the Zn(2+)-binding sequences are important for catalysis and recognition in the aminoacylation reactions of a subgroup of aminoacyl-tRNA synthetases.

Acylation↗

PubMed1988

Two-dimensional NMR analyses of dynamic structures of tRNA and the regulation of codon recognition by post-transcriptional modifications.

By two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy, we analyzed dynamic structures of various tRNA species from Escherichia coli and Bacillus subtilis. Proton resonances due to the anticodon of the tRNA molecules were unambiguously identified by NOESY and 2D-HOHAHA techniques. Thus, it was found that rigidity/flexibility of the two types of modified uridines in the first position of the anticodon were certainly related with the codon recognition properties of the tRNA species.

Anticodon↗

PubMed1979

Chemical evidence for a codon-induced allosteric change in tRNALys involving the 7-methylguanosine residue 46.

[32P]TRNALys, from Escherichia coli, was modified with kethoxal, in the presence and absence of the oligonucleotide codon (A)4. The presence of the codon resulted in a faster modification rate of the tRNA at three guanine sites which were identified by a diagonal fingerprint method. A large increase in the modification rate occurred at the 7-methylguanosine residue 46 (m7G-46) in the presence of the codon: weakly enhanced modification was observed at G-15 and G-57. It is concluded that the formation of a codon-anticodon complex induces, primarily, a conformational change involving disruption of the m7G-46 from the m7G-46 . G-22 . C-13 base triple. Subsequently, the guanines of G-15 and G-57, in the D and T loops, respectively, become slightly more reactive, suggesting a weak tendency for these two interacting arms to unfold. The results are interpreted in terms of an equilibrium between two main conformers, and a third minor one; the possible significance of these conformers in protein biosynthesis, is considered.

Base Sequence↗

PubMed1997

Chemical modification and site-directed mutagenesis of the single cysteine in motif 3 of class II Escherichia coli prolyl-tRNA synthetase.

Class II prolyl-tRNA synthetase (ProRS) from Escherichia coli contains all three of the conserved consensus motifs characteristic of class II aminoacyl-tRNA synthetases. In this study, chemical modification and site-directed mutagenesis of the single cysteine located at position 443 in motif 3 of Escherichia coli ProRS is carried out. We show that chemical modification of C443 blocks the ability of the enzyme to form the activated aminoacyl-adenylate, a prerequisite for tRNA(Pro) aminoacylation. Nearly complete protection from inactivation is achieved by preincubating the enzyme with ATP or ATP and proline, but not proline alone or tRNA(Pro). Mutagenesis of C443 to amino acids Ala, Gly, and Ser resulted in significant decreases (16-225-fold) in k(cat)/K(M)(Pro) as measured by the ATP-PP(i) exchange reaction. The Ala and Gly mutations have a relatively small effect (4-7-fold) on the overall aminoacylation reaction, while the activity of the C443S mutant in this same assay is substantially reduced (80-fold). A sequence comparison of the motif 3 region of class II synthetases shows that C443 aligns with residues that have been implicated in amino acid binding specificity. The results of our study suggest that while the thiol located at position 443 of Escherichia coli ProRS is not essential for catalysis, this residue is likely to be in a buried region that forms the prolyl-adenylate substrate binding pocket.

Amino Acid Sequence↗