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PubMed1981

Study of the interaction between yeast tRNAphe and yeast phenylalanyl-tRNA synthetase by monochromatic ultraviolet irradiation at various wavelengths. Advantages and limits of the method.

The interactions between yeast tRNAphe and phenylalanyl-tRNA synthetase were studied by analysis of the covalent adducts obtained upon monochromatic ultraviolet irradiation at different wavelengths (248, 282, 292, 302 and 313 nm). The high extent of inactivation of phenylalanyl-tRNA synthetase, together with the partial modification of tRNA, as well as the peculiar instability of most of the covalent bonds formed upon irradiation constitute severe limitations to the use of the technique and to the interpretation of the results. These disadvantages led us to select an irradiation wavelength of 248 nm and to use only mild isolation procedures allowing a good recovery of the covalent adducts formed. Seven major tryptic peptides of the enzyme were found to be cross-linked to tRNAPhe whereas six major T1-oligonucleotides were covalently linked to the protein, among these, the three cross-linked oligonucleotides previously described by Shoemaker and Schimmel (J. Biol. Chem. 250 (1975) 4440-4444) in the same system. The difference in the number of covalently linked oligonucleotides is discussed in the light of the instability of the covalent linkages. The localization of the six oligonucleotides at the inside of the two branches forming the L-shaped tRNA molecule is similar to that observed in the yeast valine system (Renaud et al., Eur. J. Biochem. 101 (1979) 475-483) and is consistent with the interaction model previously described (Rich and Schimmel, Nucl. Acids Res. 4 (1977) 1649-1665 and Ebel et al. in Transfer RNA: structure, properties and recognition, (1979) pp. 325-343 Cold Spring Harbor Laboratory, NY). The occurrence of covalent cross-linking upon irradiation in the tryptophan absorption band (302 nm) strongly suggests the participation of this residue in the stabilization of the tRNA enzyme complex.

Amino Acyl-tRNA Synthetases↗
PubMed2007

Mutations in the TPsiC loop of E. coli tRNALys,3 have varied effects on in trans complementation of HIV-1 replication.

BACKGROUND: Human immunodeficiency virus (HIV-1) exclusively selects and utilizes tRNALys,3 as the primer for initiation of reverse transcription. Several elements within the TPsiC stem loop of tRNALys,3 are postulated to be important for selection and use in reverse transcription. The post-transcriptional modification at nucleotide 58 could play a role during plus-strand synthesis to stop reverse transcriptase from re-copying the tRNA primer. Nucleotides 53 and 54 within the TPsiC stem loop of the tRNA have been shown to be important to form the complex between tRNA and the HIV-1 viral genome during initiation of reverse transcription. RESULTS: To further delineate the features of the TPsiC stem loop of tRNALys,3 in reverse transcription, we have developed a complementation system in which E. coli tRNALys,3 is provided in trans to an HIV-1 genome in which the PBS is complementary to this tRNA. Successful selection and use of E. coli tRNALys,3 results in the production of infectious virus. We have used this single round infectious system to ascertain the effects that different mutants in the TPsiC stem loop of tRNALys,3 have on complementation. Mutants were designed within the TPsiC loop (nucleotide 58) and within the stem and loop of the TPsiC loop (nucleotides 53 and 54). Analysis of the expression of E. coli tRNALys,3 mutants revealed differences in the capacity for aminoacylation, which is an indication of intracellular stability of the tRNA. Alteration of nucleotide 58 from A to U (A58U), T54G and TG5453CC all resulted in tRNALys,3 that was aminoacylated when expressed in cells, while a T54C mutation resulted in a tRNALys,3 that was not aminoacylated. Both the A58U and T54G mutated tRNALys,3 complemented HIV-1 replication similar to wild type E. coli tRNALys,3. In contrast, the TG5453CC tRNALys,3 mutant did not complement replication. CONCLUSION: The results demonstrate that post-transcriptional modification of nucleotide 58 in tRNALys,3 is not essential for HIV-1 reverse transcription. In contrast, nucleotides 53 and 54 of tRNALys,3 are important for aminoacylation and selection and use of the tRNALys,3 in reverse transcription.

Base Sequence↗
PubMed2001

Distinct modes of mature and precursor tRNA binding to Escherichia coli RNase P RNA revealed by NAIM analyses.

We have analyzed by nucleotide analog interference mapping (NAIM) pools of precursor or mature tRNA molecules, carrying a low level of Rp-RMPalphaS (R = A, G, I) or Rp-c7-deaza-RMPalphaS (R = A, G) modifications, to identify functional groups that contribute to the specific interaction with and processing efficiency by Escherichia coli RNase P RNA. The majority of interferences were found in the acceptor stem, T arm, and D arm, including the strongest effects observed at positions G19, G53, A58, and G71. In some cases (interferences at G5, G18, and G71), the affected functional groups are candidates for direct contacts with RNase P RNA. Several modifications disrupt intramolecular tertiary contacts known to stabilize the authentic tRNA fold. Such indirect interference effects were informative as well, because they allowed us to compare the structural constraints required for ptRNA processing versus product binding. Our ptRNA processing and mature tRNA binding NAIM analyses revealed overlapping but nonidentical patterns of interference effects, suggesting that substrate binding and cleavage involves binding modes or conformational states distinct from the binding mode of mature tRNA, the product of the reaction.

Anticodon↗
PubMed2000

Stabilization of the transition state for the transfer of tyrosine to tRNA(Tyr) by tyrosyl-tRNA synthetase.

Aminoacylation of tRNA(Tyr) involves two steps: (1) tyrosine activation to form the tyrosyl-adenylate intermediate; and (2) transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). In Bacillus stearothermophilus tyrosyl-tRNA synthetase, Asp78, Tyr169, and Gln173 have been shown to form hydrogen bonds with the alpha-ammonium group of the tyrosine substrate during the first step of the aminoacylation reaction. Asp194 and Gln195 stabilize the transition state complex for the first step of the reaction by hydrogen bonding with the 2'-hydroxyl group of AMP and the carboxylate oxygen atom of tyrosine, respectively. Here, the roles that Asp78, Tyr169, Gln173, Asp194, and Gln195 play in catalysis of the second step of the reaction are investigated. Pre-steady-state kinetic analyses of alanine variants at each of these positions shows that while the replacement of Gln173 by alanine does not affect the initial binding of the tRNA(Tyr) substrate, it destabilizes the transition state complex for the second step of the reaction by 2.3 kcal/mol. None of the other alanine substitutions affects either the initial binding of the tRNA(Tyr) substrate or the stability of the transition state for the second step of the aminoacylation reaction. Taken together, the results presented here and the accompanying paper are consistent with a concerted reaction mechanism for the transfer of tyrosine to tRNA(Tyr), and suggest that catalysis of the second step of tRNA(Tyr) aminoacylation involves stabilization of a transition state in which the scissile acylphosphate bond of the tyrosyl-adenylate species is strained. Cleavage of the scissile bond on the breakdown of the transition state alleviates this strain.

Acylation↗
PubMed2001

Conserved amino acids near the carboxy terminus of bacterial tyrosyl-tRNA synthetase are involved in tRNA and Tyr-AMP binding.

Bacterial tyrosyl-tRNA synthetases occur in two large subfamilies, TyrRS and TyrRZ, that possess about 25% amino acid identity. Their amino-terminal region, the active site domain, is more conserved (>36% identity). The carboxy-terminal segment of these enzymes includes the tRNA binding domain and contains only few conserved residues. Replacement of three of these residues in Acidithiobacillus ferrooxidans TyrRZ revealed that S356 and K395 play roles in tRNA binding, while H306, a residue at the junction of the catalytic and tRNA binding domains, stabilizes the Tyr-AMP:TyrRZ complex. The replacement data suggest that conserved amino acids in A. ferrooxidans TyrRZ and Bacillus stearothermophilus TyrRS play equivalent roles in enzyme function.

Adenosine Monophosphate↗
PubMed1996

A sparsomycin-resistant mutant of Halobacterium salinarium lacks a modification at nucleotide U2603 in the peptidyl transferase centre of 23 S rRNA.

Sparsomycin, a broad-spectrum antibiotic, acts at the peptidyl transferase centre of the ribosome, stabilizing peptidyl-tRNA binding at the P-site and weakening ternary complex binding. A sparsomycin-resistant mutant was isolated for the archaeon Halobacterium salinarium and shown to lack a post-transcriptional modification of U2603 (Escherichia coli numbering U2584), which is a universally conserved uridine base located within the peptidyl transferase loop of 23 S rRNA. This mutant also exhibited altered sensitivities to the peptidyl transferase antibiotics anisomycin, chloramphenicol and puromycin. Several lines of evidence indicate that the unmodified uridine base lies within the P-substrate site of the peptidyl transferase centre.

Anti-Bacterial Agents↗
PubMed1988

Inhibition of polypeptide synthesis in cell-free systems by virginiamycin S and erythromycin. Evidence for a common mode of action of type B synergimycins and 14-membered macrolides.

Macrolides, lincosamides and type B synergimycins are powerful inhibitors of protein synthesis in vivo, but many of them were found to be inactive in vitro. In the present work, we confirm that virginiamycin S (a type B synergimycin) and erythromycin (a 14-membered macrolide) have no effect on poly(U)-directed poly(Phe) synthesis. However, the amino-acid polymerization reactions directed by poly(U,G), poly(U,C), poly(A,G) and poly(A,C) were increasingly inhibited (20-50%) by both antibiotics. The action of these inhibitors proved to be template-dependent and favored by the incorporation of proline and of basic amino acids into peptides. Under these conditions, virginiamycin S and erythromycin markedly stimulated a release of peptidyl-tRNA from the ribosomes. In the poly(A,C) model system, these antibiotics produced a 50% inhibition of amino-acid incorporation into total peptides, a 70% release of ribosome-bound peptidyl-tRNA, and a 95% repression of the synthesis of long peptide chains. The production of equivalent effects at saturating concentrations of these antibiotics in the four model systems examined is suggestive of a similarity in their mode of action. Our results indicate that 14-membered macrolides and type B synergimycins can act on ribosomes during the whole elongation process. The functional block produced by both antibiotics is usually reversible, but may result in a premature release of peptidyl-tRNA when the stability of ribosomal complexes is lowered by the incorporation of basic amino acids.

Anti-Bacterial Agents↗
PubMed1995

Influence of modified nucleosides on tRNA structure as probed by two plant nucleases.

Two new enzymatic probes have been used for structural investigations of native and unmodified transcript tRNA molecules. Both probes were single-strand-specific nucleases isolated from higher plants. The results obtained after enzymatic hydrolysis of tRNAs support the earlier hypothesis that posttranscriptional modifications in tRNA help to stabilize its structure and make it more rigid.

Base Sequence↗
PubMed1984

Quantitative aspects of metal ion binding to certain transfer RNA anticodon loop modified nucleosides.

Magnesium and manganese ions bind strongly to the unusual transfer RNA anticodon loop nucleotides, N-[(9-beta-D-ribofuranosyl-9H-purin-6-yl)carbamoyl]-L-threonine 5'-monophosphate (pt6A) and uridine-5-oxyacetic acid 5'-monophosphate (pV). Potentiometric measurements have shown that the delta G for metal ion-pt6A complex formation is 2-3-times more exothermic than for AMP. Electron-nuclear longitudinal dipolar relaxation data yielded manganese-ligand atom distances which permit a three-dimensional construct of the complex in which metal is coordinated to the phosphate, carboxylate of the threonine side-chain (with the nucleotide in the anti glycosidic conformation) and N7 of the adenine ring. Similarly, manganese binds strongly to pV, involving phosphate and carboxylate functions. It is possible that a facet of the functional role of these unusual residues is to chelate magnesium ions and in so doing permit optimum anticodon loop conformational stability and stability of tRNA-mRNA-ribosome complexes.

Anticodon↗
PubMed2002

Uncoupling of initiation factor eIF5B/IF2 GTPase and translational activities by mutations that lower ribosome affinity.

Translation initiation factor eIF5B/IF2 is a GTPase that promotes ribosomal subunit joining. We show that eIF5B mutations in Switch I, an element conserved in all GTP binding domains, impair GTP hydrolysis and general translation but not eIF5B subunit joining function. Intragenic suppressors of the Switch I mutation restore general translation, but not eIF5B GTPase activity. These suppressor mutations reduce the ribosome affinity of eIF5B and increase AUG skipping/leaky scanning. The uncoupling of translation and eIF5B GTPase activity suggests a regulatory rather than mechanical function for eIF5B GTP hydrolysis in translation initiation. The translational defect suggests eIF5B stabilizes Met-tRNA(i)(Met) binding and that GTP hydrolysis by eIF5B is a checkpoint monitoring 80S ribosome assembly in the final step of translation initiation.

Alleles↗
PubMed1982

Conservation of secondary structure in 5 S ribosomal RNA: a uniform model for eukaryotic, eubacterial, archaebacterial and organelle sequences is energetically favourable.

The most commonly accepted secondary structure models for 5S RNA differ for molecules of eubacterial origin, where the four-helix model of Fox and Woese is generally cited, and those of eukaryotic origin, where a fifth helix is assumed to exist. We have carefully aligned all available sequences from eukaryotes, eubacteria, chloroplasts, archaebacteria and plant mitochondria. We could thus derive a unified secondary structure model applicable to all 5S RNA sequences known to-date. It contains the five helices already present in the eukaryotic model, extended by additional segments that were not previously assumed to be universally present. One of the helices can be written in two equilibrium forms, which could reflect the existence of a flexible, dynamic structure. For the derivation of the model and the estimation of the free energies we followed a set of rules optimized to predict the tRNA cloverleaf. The stability of the unified model is higher than that of nearly all previously proposed sequence-specific and general models.

Animals↗
PubMed2006

The antibiotic kasugamycin mimics mRNA nucleotides to destabilize tRNA binding and inhibit canonical translation initiation.

Kasugamycin (Ksg) specifically inhibits translation initiation of canonical but not of leaderless messenger RNAs. Ksg inhibition is thought to occur by direct competition with initiator transfer RNA. The 3.35-A structure of Ksg bound to the 30S ribosomal subunit presented here provides a structural description of two Ksg-binding sites as well as a basis for understanding Ksg resistance. Notably, neither binding position overlaps with P-site tRNA; instead, Ksg mimics codon nucleotides at the P and E sites by binding within the path of the mRNA. Coupled with biochemical experiments, our results suggest that Ksg indirectly inhibits P-site tRNA binding through perturbation of the mRNA-tRNA codon-anticodon interaction during 30S canonical initiation. In contrast, for 70S-type initiation on leaderless mRNA, the overlap between mRNA and Ksg is reduced and the binding of tRNA is further stabilized by the presence of the 50S subunit, minimizing Ksg efficacy.

Aminoglycosides↗
PubMed1995

Protein synthesis initiation factor eIF-1A is a moderately abundant RNA-binding protein.

Eukaryotic initiation factor (eIF) 1A (formerly called eIF-4C) is a small protein that promotes dissociation of 80 S ribosomes into subunits, stabilizes methionyl-tRNA binding to 40 S ribosomal subunits, and is required for the binding of mRNA to ribosomes. The sequence of eIF-1A derived from its cloned cDNA possesses a high frequency of basic residues and acidic residues at its N and C termini, respectively. Northwestern blotting with a fragment of mRNA indicates that eIF-1A binds RNA. Overexpression of the human eIF-1A cDNA in Escherichia coli and subsequent purification enabled us to prepare large quantities of active factor. The level of eIF-1A in HeLa cells determined by Western immunoblotting is 0.01% of total protein, which corresponds to 0.2 molecules of eIF-1A/ribosome. The moderate abundance means that eIF-1A is equal to or in excess of native 40 S subunits and suggests that the factor may not be limiting for protein synthesis, a conclusion reinforced by the failure of overproduced eIF-1A to stimulate translation rates in transiently transfected COS-1 cells. S1 nuclease protection and primer extension analyses show that eIF-1A mRNA possesses an unusually long 5'-untranslated leader that is very G/C-rich (72%). Unexpectedly, the mRNA is efficiently translated in HeLa cells as judged by polysome profile analyses.

Animals↗
PubMed2002

NMR analysis of bovine tRNATrp: conformation dependence of Mg2+ binding.

NMR was used to study the solution structure of bovine tRNA(Trp) hyperexpressed in Escherichia coli. With the use of (15)N labeling and site-directed mutagenesis to assign overlapping resonances through the base pair replacement of U(71)A(2) by G(2)C(71), U(27)A(43) by G(27)C(43), and G(12)C(23) by U(12)A(23), the resonances of all 26 observable imino protons in the helical regions and in the tertiary interactions were assigned unambiguously by means of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear single quantum coherence methods. When the discriminator base A(73) and the G(12)C(23) base pair on the D stem, two identity elements on bovine tRNA(Trp) that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G(73) and U(12)A(23), respectively, NMR analysis revealed an important conformational change in the U(12)A(23) mutant but not in the G(73) mutant molecule. Thus A(73) appears to be directly recognized by tryptophanyl-tRNA synthetase, and G(12)C(23) represents an important structural determinant. Mg(2+) effects on the assigned resonances of imino protons allowed the identification of strong, medium, and weak Mg(2+) binding sites in tRNA(Trp). Strong Mg(2+) binding modes were associated with the residues G(7), s(4)U(8) (where s(4)U is 4-thiouridine), G(12), and U(52). The observations that G(42) was associated with strong Mg(2+) binding in only the U(12)A(23) mutant tRNA(Trp) but not the wild type or G(73) mutant tRNA(Trp) and that the G(7), s(4)U(8), G(24), and G(22) imino protons are associated with a two-site Mg(2+) binding mode in wild type and G(73) mutant but only a one-site mode in the U(12)A(23) mutant established the occurrence of conformational change in the U(12)A(23) mutant tRNA(Trp). These observations also established the dependence of Mg(2+) binding on tRNA conformation and the usefulness of Mg(2+) binding sites as conformational probes. The thermal titration of tRNA(Trp) in the presence and absence of 10 mm Mg(2+) indicated that overall tRNA(Trp) structure stability was increased by more than 15 degrees C by the presence of Mg(2+).

Animals↗
PubMed2005

Naturally occurring polyamines: interaction with macromolecules.

The naturally occurring polyamines, spermine [NH2(CH2)3NH(CH2)4NH(CH2)3NH2] and spermidine [NH2(CH2)3NH(CH2)4NH2], as well as the diamine putrescine [NH2(CH2)4NH2], are widely spread in nature. They occur in plants, micro-organisms and animal tissues and fulfil many important physiological functions. Due to their cationic nature they interact with negatively charged macromolecules such nucleic acids, phospholipids and proteins. This ionic interaction, which is reversible, leads to the stabilization of DNA, tRNA, membranes and some proteins. Early studies demonstrated that polyamines stimulate the growth of pro- and eukaryotic cells and that they play an important role in carcinogenesis and in malignant transformation processes. As a result of these studies various inhibitors of polyamine biosynthesis have been synthesized and are used to combat cancer and parasitic diseases (e.g., African sleeping sickness).

Animals↗
PubMedDate not supplied

[The effect of modification of nucleotide-37 on the interaction of aminoacyl-tRNA with the A-site of the 70S ribosome].

To estimate the effect of modified nucleotide-37, the interaction of two yeast aminoacyl-tRNAs (Phe-tRNAK+YPhe and Phe-tRNAK-YPhe) with the A site of complex [70S.poly(U).deacylated tRNA(Phe) in the P site] was assayed at 0-20 degrees C. As comparisons with native Phe-tRNAK+YPhe showed, removal of the Y base decreased the association constant of Phe-tRNAK-YPhe and the complex by an order of magnitude at any temperature, and increased the enthalpy of their interaction by 23 kJ/mol. When the Y base was present in the anticodon loop of deacylated tRNA(Phe) bound to the P site of the 70S ribosome, twice higher affinity for the A site was observed for Phe-tRNAK-YPhe but not for Phe-tRNAK+YPhe. Thus, the modified nucleotide 3' of the Phe-tRNA(Phe) anticodon stabilized the codon-anticodon interaction both in the A and in the P sites of the 70S ribosome.

Anticodon↗
PubMed1990

Recognition of tRNA(Tyr) by tyrosyl-tRNA synthetase.

In this review, I have brought together and compared the available data on the interaction between tRNA(Tyr) and tyrosyl-tRNA synthetases (TyrTS) of prokaryotic origins. The amino acid sequences of the heterologous TyrTS that can charge Escherichia coli tRNA(Tyr), show that the residues involved in the binding and recognition of tyrosine are strictly conserved whereas those involved in the interaction with tRNA(Tyr) are only weakly similar. The results of in vivo genetic complementation experiments indicate that the identity elements of tRNAs and the recognition mechanisms of such elements by the synthetases have been conserved during evolution. Heterologous or mutant tRNA(Tyr) are quantitatively charged by E coli TyrTS; the set of their common residues contains less than 10 elements if one excludes the invariant and semi-invariant residues of tRNAs. The residues of this set are candidates for a specific recognition by TyrTS. So far, adenosine-73 is the only residue for which a specific recognition of the base has been demonstrated. The residues that might serve as identity elements for E coli tRNA(Tyr) [McClain WH, Nicholas Jr HB (1987) J Mol Biol 194, 635-642] do not belong to the above set of conserved residues and therefore probably play negative roles, enabling tRNA(Tyr) to avoid non-cognate synthetases. Comparison of the charging and stability properties of mutant tRNA(Tyr) su +3 shows that bases 1 and 72 must pair (either by Watson-Crick or non-canonical hydrogen bonds) and adopt a geometry which is compatible with the helical structure of the acceptor stem in order for the mutant tRNA(Tyr) to be charged with tyrosine. If bases 1 and 72 or bases 2 and 71 cannot form such pairings, the suppressor phenotype of the mutant tRNA(Tyr)su +3 becomes thermosensitive. The weakening of base pair 1/72 by mutation or the change of adenosine-73 into guanosine results in the charging of tRNA(Tyr)su +3 with glutamine. Comparison of the structural model of the TyrTS/tRNA(Tyr) complex with the crystallographic structure of the GlnTS/tRNA(Gln) complex indicates that the mechanisms for the recognition of the acceptor arm are different in the 2 cases. Chemical attack and molecular modeling experiments have indicated that the acceptor end of tRNA(Tyr) ... CCCA3'-OH, remains mobile after the initial binding of tRNA(Tyr) to TyrTS.

Base Sequence↗
PubMedDate not supplied

[The effect of modification of tRNA nucleotide-37 on the tRNA interaction with the P- and A-site of the 70S ribosome Escherichia coli].

A modified nucleotide on the 3'-side of the anticodon loop of tRNA is one of the most important structure element regulating codon-anticodone interaction on the ribosome owing to the stacking interaction with the stack of codon-anticodon bases. The presence and identity (pyrimidine, purine or modified purine) of this nucleotide has an essential influence on the energy of the stacking interaction on A- and P-sites of the ribosome. There is a significant influence of the 37-modification by itself on the P-site, whereas there is no such one on the A-site of the ribosome. Comparison of binding enthalpies of tRNA interactions on the P- or A-site of the ribosome with the binding enthalpies of the complex of two tRNAs with the complementary anticodones suggests that the ribosome by itself significantly endows in the thermodynamics of codon-anticodon complex formation. It happens by additional ribosomal interactions with the molecule of tRNA or indirectly by the stabilization of codon-anticodon conformation. In addition to the stacking, tRNA binding in the A and P sites is futher stabilized by the interactions involving some magnesium ions. The number of them involved in those interactions strongly depends on the nucleotide identity in the 37-position of tRNA anticodon loop.

Acetylation↗