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PubMed1971

Interaction between the first enzyme for histidine biosynthesis and histidyl transfer ribonucleic acid.

Previous studies suggested that phosphoribosyltransferase, which catalyzes the first step of the pathway for histidine biosynthesis in Salmonella typhimurium and which is sensitive to inhibition by histidine, plays a role in repression of the histidine operon. Recently, we showed that the enzyme has a high affinity for histidyl transfer ribonucleic acid (His-tRNA), which is known to participate in the repression process. In the present study, we have investigated further the interaction between the enzyme and His-tRNA. We found that His-tRNA binds at a site on phosphoribosyltransferase distinct from the catalytic site and the histidine-sensitive site; that the substrates of the enzyme inhibit the binding of His-tRNA, whereas histidine does not do so; that, once a complex has been formed between phosphoribosyltransferase and His-tRNA, the substrates of the enzyme decrease the stability of the complex, whereas histidine is without effect; and that purified phosphoribosyltransferase which has a defect in its inhibition by histidine (produced by mutation) displays an altered ability to bind His-tRNA, a finding which may be a reflection of the fact that mutants producing such a defective enzyme display an alteration of the repression process.

Acrylates↗
PubMedDate not supplied

Stabilization of the ternary complex EF-Tu.GTP.valyl-tRNAval by ammonium salts.

In a search for crystallizing conditions for the ternary complex EF-Tu.GTP.valyl-tRNAval, the influence of various salts on its stability has been examined by measuring the rate of deacylation of the aminoacyl-tRNA in the complex. The most striking result is the general higher stability in solutions of ammonium salts and, in particular, the enhancement of this effect by sulphate and citrate. Thus sodium sulphate and citrate lead to destabilization of the complex, as expected from conventional considerations of adding salt, whereas the corresponding ammonium salts stabilize the complex as shown, for example, by an increase in the half-life of the valyl-tRNAval in the complex from about 20 hours to at least 300 hours in the presence of 1.2 M ammonium sulphate. These results suggest that ammonium sulphate and ammonium citrate might be very suitable precipitants for crystallization studies of the ternary complex.

Crystallization↗
PubMed2003

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

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

Acylation↗
PubMed2006

Ribonucleome analysis identified enzyme genes responsible for wybutosine synthesis.

Wybutosine (yW) is a hyper modified guanosine with a tricyclic base found at the 3'-position adjacent to the anticodon of eukaryotic phenylalanine tRNA. yW supports reading frame maintenance by stabilizing codon-anticodon interactions during decoding on the ribosome. To identify genes responsible for yW synthesis from uncharacterized genes of Saccharomyces cerevisiae, we employed a systematic reverse genetic approach combined with mass spectrometry ('ribonucleome analysis'). We found four new genes YPL207w, YML005w, YOL141w and YGL050w (named TYW1, TYW2, TYW3 and TYW4, respectively) to be responsible for wybutosine (yW) synthesis. Mass spectrometric analysis of each modification intermediate of yW revealed its sequential biosynthetic pathway. Multi-step enzymatic formation of yW was partially reconstituted in vitro using recombinant proteins. The results suggested that yW synthesis might proceed through sequential reactions in a complex formed by multiple protein components assembled with the precursor tRNA.

Gene Deletion↗
PubMed1976

Specificity and properties of the destabilization, induced by initiation factor IF-3, of ternary complexes of the 30-S ribosomal subunit, aminoacyl-tRNA and polynucleotides.

Initiation factor IF-3 causes the destabilization of preformed ternary complexes of 30-S ribosomal subunit, codons and aminoacyl-tRNAs or peptidyl-tRNA. This destabilization is dilution-dependent and affects all ternary complexes with the exception of those containing the initiator fMet-tRNA, which remain more resistant to IF-3-induced destabilization under the various conditions studied. Several possible reasons for this specificity have been examined. It was found that the basis for the specificity is not: (a) an intrinsic greater stability of the ternary complexes containing fMet-tRNA, (b) the amoung of aminoacyl-tRNA bound to the ribosome, (c) the conditions under which the ternary complex is made or (d) the formylation of the amino group. On the other hand, the nature of the polynucleotide in response to which the ternary complex is formed was found to influence the amount of aminoacyl-tRan bound to the ribosome, and to some extent the amount of aminoacyl-tRNA which can be relased. The ternary complex containing the mischarged initiator tRNA fVal-tRNAfMet displays greater resistance to the IF-3-induced destabilization than the complex containing fVal-tRNAVal. These results indicate that the specificity of the IF-3 activity is due to the special structural feature of the initiator tRNA molecule and to some extent to the nature of the polynucleotide. The IF-3-induced destabilization of ternary complexes was found to be little affected by variations in reaction conditons, so that this IF-3 activity can be used to measure the stoichiometric binding of IF-3 to the ribosome over a broad range of pH and K+ and Mg2+ concentrations. Several antibiotics have been tested for their capacity to interfere with this reaction; only high concentrations of tetracycline blocked this IF-3 activity.

Anti-Bacterial Agents↗
PubMed1979

Methionyl-tRNA-Met-f deacylase. Purification, characterization, and effects on translational initiation complexes.

A methionyl-tRNA-Met-f deacylase was found in ribosomal salt wash from cultured human cells of the HeLa line. This enzyme was purified by the use of DEAE-cellulose, ammonium sulfate precipitation, gel filtration and isoelectric focusing, and appears to be a protein with a native molecular weight of 80,000, which consists of two 40,000-Mr subunits. The mechanism of the Met-tRNA-Met-f deacylase is shown to involve end-product inhibition by the deacylated form of Met-tRNA-Met-f. The methionyl-tRNA-Met-f deacylase is rather specific for Met-tRNA-Met-f as opposed to Met-tRNA-Met-m, has a KCl optimum of 85 mM, is inhibited by MgCl2 and is inhibited by GTP and NAD+ at physiological concentration. 40-S and 60-S subunits inhibit the enzyme, possibly by binding to it. The stability of translational initiation complexes, containing methionyl-tRNA-Met-f, was investigated in the presence of the enzyme. Purified ternary complex was slowly broken down by the enzyme, while the 40-S-subunit . Met-tRNA-Met-f complex was stable in the presence of enzyme. The 80-S complex formed with A-U-G trinucleotide as the message molecule was broken down, whereas the 80-S complex formed with globin mRNA was stable in the presence of the enzyme. The physiological role of this enzyme is unclear, but it might act to regulate initiation by deacylating Met-tRNA-Met-f.

Acyltransferases↗
PubMed1999

A mutant HemA protein with positive charge close to the N terminus is stabilized against heme-regulated proteolysis in Salmonella typhimurium.

The HemA enzyme (glutamyl-tRNA reductase) catalyzes the first committed step in heme biosynthesis in the enteric bacteria. HemA is mainly regulated by conditional protein stability; it is stable and, consequently, more abundant in heme-limited cells but unstable and less abundant in normally growing cells. Both the Lon and ClpAP energy-dependent proteases contribute to HemA turnover in vivo. Here we report that the addition of two positively charged lysine residues to the third and fourth positions at the HemA N terminus resulted in complete stabilization of the protein. By contrast, the addition of an N-terminal myc epitope tag did not affect turnover. This result confirms the importance of the N-terminal sequence for proteolysis of HemA. This region of the protein also contains a proline flanked by hydrophobic residues, a motif that has been suggested to be important for Lon-mediated proteolysis of UmuD. However, mutation of this motif did not affect the turnover of HemA protein. Cells expressing the stabilized HemA[KK] mutant protein display substantial defects in heme regulation.

ATP-Dependent Proteases↗
PubMed2002

Potassium functionally replaces the second lysine of the KMSKS signature sequence in human tyrosyl-tRNA synthetase.

Unlike their bacterial homologues, a number of eukaryotic tyrosyl-tRNA synthetases require potassium to catalyze the aminoacylation reaction. In addition, the second lysine in the class I-specific KMSKS signature motif is absent from all known eukaryotic tyrosyl-tRNA synthetase sequences, except those of higher plants. This lysine, which is the most highly conserved residue in the class I aminoacyl-tRNA synthetase family, has been shown to interact with the pyrophosphate moiety of the ATP substrate in the Bacillus stearothermophilus tyrosyl-tRNA synthetase. Equilibrium dialysis and pre-steady-state kinetic analyses were used to determine the role that potassium plays in the tyrosine activation reaction in the human tyrosyl-tRNA synthetase and whether it can be replaced by any of the other alkali metals. Kinetic analyses indicate that potassium interacts with the pyrophosphate moiety of ATP, stabilizing the E.Tyr.ATP and E.[Tyr-ATP] complexes by 2.3 and 4.3 kcal/mol, respectively. Potassium also appears to stabilize the asymmetric conformation of the human tyrosyl-tRNA synthetase dimer by 0.7 kcal/mol. Rubidium is the only other alkali metal that can replace potassium in catalyzing tyrosine activation, although the forward rate constant is half of that observed when potassium is present. The above results are consistent with the hypothesis that potassium functionally replaces the second lysine in the KMSKS signature sequence. Possible implications of these results with respect to the design of antibiotics that target bacterial aminoacyl-tRNA synthetases are discussed.

Amino Acid Motifs↗
PubMed2005

Structural insights into translational fidelity.

The underlying basis for the accuracy of protein synthesis has been the subject of over four decades of investigation. Recent biochemical and structural data make it possible to understand at least in outline the structural basis for tRNA selection, in which codon recognition by cognate tRNA results in the hydrolysis of GTP by EF-Tu over 75 A away. The ribosome recognizes the geometry of codon-anticodon base pairing at the first two positions but monitors the third, or wobble position, less stringently. Part of the additional binding energy of cognate tRNA is used to induce conformational changes in the ribosome that stabilize a transition state for GTP hydrolysis by EF-Tu and subsequently result in accelerated accommodation of tRNA into the peptidyl transferase center. The transition state for GTP hydrolysis is characterized, among other things, by a distorted tRNA. This picture explains a large body of data on the effect of antibiotics and mutations on translational fidelity. However, many fundamental questions remain, such as the mechanism of activation of GTP hydrolysis by EF-Tu, and the relationship between decoding and frameshifting.

Anti-Bacterial Agents↗
PubMed1982

Purification and properties of chloroplast leucyl-tRNA synthetase from a higher plant: Phaseolus vulgaris.

The chloroplast leucyl-tRNA synthetase from Phaseolus vulgaris was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, hydroxylapatite, and phosphocellulose. Finally, the pure enzyme was obtained after affinity chromatography on Blue Sepharose CL-6B using specific elution with a pure Escherichia coli tRNALeu isoacceptor. The specific activity of chloroplast leucyl-tRNA synthetase (1550 units/mg) is the highest ever obtained for a higher plant aminoacyl-tRNA synthetase. The purified enzyme has an optimal pH of 9.0 and Km values are, respectively, 2.6 X 10(-6) M for unfractionated E. coli tRNA, 0.85 X 10(-6) M for E. coli tRNA5Leu, 1.8 X 10(-4) M for ATP, and 1.4 X 10(-5) M for L-leucine. Chloroplast leucyl-tRNA synthetase is a large monomer which has a Mr of 122,000 as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate and urea and by gel filtration. Determination of Stokes radius, diffusion coefficient, and frictional ratio suggests that the enzyme structure is rather compact. The amino acid composition shows a relatively large proportion of apolar residues. Specific antibodies were raised in rabbits against the pure chloroplast leucyl-tRNA synthetase.

Amino Acids↗
PubMed2001

Codon bias at the 3'-side of the initiation codon is correlated with translation initiation efficiency in Escherichia coli.

The codon that follows the AUG initiation triplet (+2 codon) affects gene expression in Escherichia coli. We have extended this analysis using two model genes lacking any apparent Shine-Dalgarno sequence. Depending on the identity of the +2 codon a difference in gene expression up to 20-fold could be obtained. The effects did not correlate with the levels of intracellular pools of cognate tRNA for the +2 codon, with putative secondary mRNA structures, or with mRNA stability. However, most +2 iso-codons that were decoded by the same species of tRNA gave pairwise similar effects, suggesting that the effect on gene expression was associated with the decoding tRNA. High adenine content of the +2 codon was associated with high gene expression. Of the fourteen +2 codons that mediated the highest efficiency, all except two had an adenine as the first base of the codon. Analysis of the 3540 E. coli genes from the TransTerm database revealed that codons associated with high gene expression in the two expression systems are over-represented at the +2 position in natural genes. Codons that are associated with low gene expression are under-represented. The data suggest that evolution has favored codons at the +2 position that give high translation initiation.

Codon↗
PubMed1991

Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA.

Oligonucleotide fragments derived from the 3' CCA terminus of acylated tRNA, such as CACCA-(AcPhe), UACCA-(AcLeu), and CAACCA-(fMet), bind specifically to ribosomes in the presence of sparsomycin and methanol [Monro, R. E., Celma, M. L. & Vazquez, D. (1969) Nature (London) 222, 356-358]. All three oligonucleotides protect a characteristic set of bases in 23S rRNA from chemical probes: G2252, G2253, A2439, A2451, U2506, and U2585. A2602 shows enhanced reactivity. These account for most of the same bases that are protected when peptidyl-tRNA analogues such as AcPhe-tRNA are bound to the ribosomal P site, and correspond precisely to those bases whose protection is abolished by removal of the 3'-CA end of tRNA. We conclude that most of the observed interactions between tRNA and 23S rRNA in the 50S ribosomal P site involve the conserved CCA terminus of tRNA. Sparsomycin may inhibit protein synthesis by stabilizing interaction between the peptidyl-CCA and the 23S P site, preventing formation of the intermediate A/P hybrid state.

Base Sequence↗
PubMed2000

tRNA recognition of tRNA-guanine transglycosylase from a hyperthermophilic archaeon, Pyrococcus horikoshii.

In the biosynthesis of archaeosine, archaeal tRNA-guanine transglycosylase (TGT) catalyzes the replacement of guanine at position 15 in the D loop of most tRNAs by a free precursor base. We examined the tRNA recognition of TGT from a hyperthermophilic archaeon, Pyrococcus horikoshii. Mutational studies using variant tRNA(Val) transcripts revealed that both guanine and its location (position 15) were strictly recognized by TGT without any other sequence-specific requirements. It appeared that neither the global L-shaped structure of a tRNA nor the local conformation of the D loop contributed to recognition by TGT. A minihelix composed of the acceptor stem and D arm of tRNA(Val), designed as a potential minimal substrate, failed to serve as a substrate for TGT. Only a minihelix with mismatched nucleotides at the junction between the two domains served as a good substrate, suggesting that mismatched nucleotides in the helix provide the specific information that allows TGT to recognize the guanine in the D loop. Our findings indicate that the tRNA recognition requirements of P. horikoshii TGT are sufficiently limited and specific to allow the enzyme to recognize efficiently any tRNA species whose structure is not fully stabilized in an extremely high temperature environment.

Anticodon↗
PubMed2002

Combinatorial properties of RNA secondary structures.

The secondary structure of an RNA molecule is of great importance and possesses influence, e.g., on the interaction of tRNA molecules with proteins or on the stabilization of mRNA molecules. The classification of secondary structures by means of their order proved useful with respect to numerous applications. In 1978, Waterman, who gave the first precise formal framework for the topic, suggested to determine the number a(n,p) of secondary structures of size n and given order p. Since then, no satisfactory result has been found. Based on an observation due to Viennot et al., we will derive generating functions for the secondary structures of order p from generating functions for binary tree structures with Horton-Strahler number p. These generating functions enable us to compute a precise asymptotic equivalent for a(n,p). Furthermore, we will determine the related number of structures when the number of unpaired bases shows up as an additional parameter. Our approach proves to be general enough to compute the average order of a secondary structure together with all the r-th moments and to enumerate substructures such as hairpins or bulges in dependence on the order of the secondary structures considered.

Algorithms↗
PubMed1998

The structure of HIV-1 reverse transcriptase complexed with an RNA pseudoknot inhibitor.

Small RNA pseudoknots, selected to bind human immunodeficiency virus type 1 (HIV-1) reverse transcriptase tightly, are potent inhibitors of reverse transcriptase. The co-crystal structure of reverse transcriptase complexed with a 33 nucleotide RNA pseudoknot has been determined by fitting the ligand into a high quality, 4-fold averaged 4.8 A resolution electron density map. The RNA is kinked between stems S1 and S2, thereby optimizing its contacts with subunits of the heterodimer. Its binding site extends along the cleft that lies between the polymerase and RNase H active sites, partially overlaps with that observed for duplex DNA and presumably overlaps some portion of the tRNA site. Stem S2 and loop L1 stabilize the 'closed' conformation of the polymerase through extensive electrostatic interactions with several basic residues in helix I of the p66 thumb and in the p66 fingers domain. Presumably, this RNA ligand inhibits reverse transcriptase by binding to a site that partly overlaps the primer-template binding site.

Crystallization↗
PubMed1995

Replication of avian leukosis viruses with mutations at the primer binding site: use of alternative tRNAs as primers.

We have tested whether avian leukosis viruses (ALVs) can use tRNAs other than tRNATrp to initiate reverse transcription. The primer binding site (PBS) of a wild-type ALV provirus, which is complementary to the 3' end of tRNA(Trp), was replaced with sequences homologous to the 3' ends of six different chicken tRNAs (tRN(APro), tRNA(Lys), tRNA(Met), tRNA(Ile), tRNA(Phe), and tRNA(Ser)). Transfection of these proviruses into chicken embryo fibroblasts resulted in the production of infectious viruses, all of which apparently used the tRNA specified by the mutated PBS to replicate. However, growth of these viruses resulted in reversion to the wild-type (tRNA(Trp)) PBS. Some of the viruses revert quite quickly, while others are more stable. The relative stability of a given PBS correlated with the concentration of the corresponding tRNA in the virion. We determined the percentage of viral RNA that had a tRNA bound to the PBS and found that the occupancy rate is lower in the mutants than in the wild-type virus. We conclude that many different tRNAs can be used as primers to initiate reverse transcription in ALV. However, ALVs that use tRNA(Trp) have a growth advantage over ALVs that use other tRNAs.

Animals↗
PubMed1990

Conformational changes of aminoacyl-tRNA and uncharged tRNA upon complex formation with polypeptide chain elongation factor Tu.

The conformation change of Thermus thermophilus tRNA(1Ile) upon complex formation with T. thermophilus elongation factor Tu (EF-Tu) was studied by analysis of the circular dichroism (CD) bands at 315 nm (due to the 2-thioribothymidine residue in the T-loop) and at 295 nm (due to the core structure of tRNA). Formation of the ternary complex of isoleucyl-tRNA(1Ile) and EF-Tu.GTP increased the intensities of these CD bands, indicating stabilization of the association between the T-loop and the D-loop and also a significant conformation change of the core region. Upon complex formation of EF-Tu.GTP and uncharged tRNA, however, the conformation of the core region is not changed, while the association of the two loops is still stabilized. On the other hand, the binding with EF-Tu.GDP does not appreciably affect the conformation of isoleucyl-tRNA or uncharged tRNA. These indicate the importance of the gamma-phosphate group of GTP and the aminoacyl group in the formation of the active complex of aminoacyl-tRNA and EF-Tu.GTP.

Circular Dichroism↗