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At least 145 records · Page 8Linked to original sources
PubMed1989

The modulatory element TNNCT affects transcription of a Drosophila tRNA(Val)4 gene without affecting transcription complex stability.

We have previously identified a sequence of the general form TNNCT in the 5'-flanking region of a Drosophila melanogaster tRNA(Val)4 gene which positively modulates in vitro transcription. The pentadeoxynucleotide is also present in the 5'-flank of other Drosophila tRNA genes which direct transcription efficiently in vitro. We have examined transcription of a mutant of this gene further to understand the role of the TNNCT sequence. When template competition experiments were carried out, a template which contained mutations in TNNCT was a better competitor than was the wildtype template. The time-course of transcription and the effects of temperature and ionic strength on transcription indicated that mutating the TNNCT did not alter the efficiency of stable complex formation. It is proposed that the pentadeoxynucleotide TNNCT may influence the rate of transcription initiation by RNA polymerase III bound to transcription complexes.

Animals↗
PubMed1982

Stabilization of the tertiary structure of yeast phenylalanine tRNA by [Co(NH3)6]3+. X-ray evidence for hydrogen bonding to pairs of guanine bases in the major groove.

The sites of three [Co(NH3)6]3+ ions bound to the phenylalanine tRNA of yeast have been determined by X-ray diffraction analysis. [Co(NH3)6]3+ binds to purine-purine sequences in yeast tRNA Phe. It is different from the binding fo Co2+, which binds to the base and phosphate of residue G15. There are no direct metal-nucleotide bonds, although hydrogen bonding of the coordinated ammines to double-helical guanylguanosine sequences in the major groove and to phosphate oxygen in neighboring polynucleotide strands increases the stability of the structure. Hydrogen-bonding appears to be via cis ammine ligands to N(7) and O(6) positions of adjacent purine bases.

Amino Acyl-tRNA Synthetases↗
PubMed1993

Accuracy of tRNA charging and codon: anticodon recognition; relative importance for cellular stability.

Cellular homeostasis and the mechanisms which control homeostasis are important for understanding such fundamental processes as ageing and the origin of life. Several models have studied the importance of accurate protein synthesis for cellular stability, but these models have not considered the complexities of the translation process in any detail. Here we develop a new model which describes the interplay between aminoacyl-tRNA (aatRNA) synthetases, the cellular pool of charged tRNAs and the process of codon: anticodon recognition. We also take the processive character of the ribosomes into account. In common with previous work, our model predicts that the cellular translation apparatus can either be stable or deteriorate progressively with time. However, because our model explicitly describes different subreactions of the overall translation process, we are also able to assess the relative importance of accurate tRNA charging and codon: anticodon recognition for cellular stability. It appears that the tRNA charging by the aatRNA synthetases plays the key role in controlling the long-term stability of the cell. Ribosomal errors are less important because error-prone ribosomes, being processive, produce mainly inactive proteins which do not contribute to error propagation within the translation machinery.

Amino Acyl-tRNA Synthetases↗
PubMed1987

[Isolation, polypeptide composition and properties of aminoacyl-tRNA-synthetase complexes from the rabbit liver].

The procedure for isolation of the aminoacyl-tRNA-synthetase complex from rabbit liver based on the affinity chromatography on heparin- and tRNA-Sepharose has been developed. The complex has a Mr of about 1100 kD and is made up of 10 polypeptides, eight of which are aminoacyl-tRNA synthetase. The complex stability was studied under various conditions. The data obtained are discussed in terms of the involvement of hydrophobic domains of aminoacyl-tRNA synthetases in the stabilization of the complex.

Amino Acyl-tRNA Synthetases↗
PubMed1987

Site-directed mutagenesis reveals transition-state stabilization as a general catalytic mechanism for aminoacyl-tRNA synthetases.

Some aminoacyl-tRNA synthetases of almost negligible homology do have a small region of similarity around four-residue sequence His-Ile(or Leu or Met)-Gly-His(or Asn), the HIGH sequence. The first histidine in this sequence in the tyrosyl-tRNA synthetase, His-45, has been shown to form part of a binding site for the gamma-phosphate of ATP in the transition state for the reaction as does Thr-40. Residue His-56 in the valyl-tRNA synthetase begins a HIGH sequence, and there is a threonine at position 52, one position closer to the histidine than in the tyrosyl-tRNA synthetase. The mutants Thr----Ala-52 and His----Asn-56 have been made and their complete free energy profiles for the formation of valyl adenylate determined. Difference energy diagrams have been constructed by comparison with the reaction of wild-type enzyme. The difference energy profiles are very similar to those for the mutants Thr----Ala-40 and His----Asn-45 of the tyrosyl-tRNA synthetase. Thr-52 and His-56 of the valyl-tRNA synthetase contribute little binding energy to valine, ATP, and Val-AMP. Instead, the wild-type enzyme binds the transition state and pyrophosphate some 6 kcal/mol more tightly than do the mutants. Preferential transition-state stabilization is thus an important component of catalysis by the valyl-tRNA synthetase. Further, by analogy to the tyrosyl-tRNA synthetase, the valyl-tRNA synthetase has a binding site for the gamma-phosphate of ATP in the transition state, and this is likely to be a general feature of aminoacyl-tRNA synthetases that have a HIGH region.

Amino Acid Sequence↗
PubMed1999

Conditional stability of the HemA protein (glutamyl-tRNA reductase) regulates heme biosynthesis in Salmonella typhimurium.

In many bacteria, including the enteric species Salmonella typhimurium and Escherichia coli, heme is synthesized starting from glutamate by a pathway in which the first committed step is catalyzed by the hemA gene product, glutamyl-tRNA reductase (HemA). We have demonstrated previously that when heme limitation is imposed on cultures of S. typhimurium, HemA enzyme activity is increased 10- to 25-fold. Western (immunoblot) analysis with monoclonal antibodies reactive with HemA revealed that heme limitation results in a corresponding increase in the abundance of the enzyme. Similar regulation was also observed for E. coli. The near absence of regulation of hemA-lac operon fusions suggested a posttranscriptional control. We report here the results of pulse-labeling and immunoprecipitation studies of this regulation. The principal mechanism that contributes to elevated HemA abundance is protein stabilization. The half-life of HemA protein is approximately 20 min in unrestricted cells but increases to >300 min in heme-limited cells. Similar regulation was observed for a HemA-LacZ hybrid protein containing almost all of the HemA protein (416 residues). Sodium azide prevents HemA turnover in vivo, suggesting a role for energy-dependent proteolysis. This was confirmed by the finding that HemA turnover is completely blocked in a lon clpP double mutant of E. coli. Each single mutant shows only a small effect. The ClpA chaperone, but not ClpX, is required for ClpP-dependent HemA turnover. A hybrid HemA-LacZ protein containing just 18 amino acids from HemA is also stabilized in the lon clpP double mutant, but this shorter fusion protein is not correctly regulated by heme limitation. We suggest that the 18 N-terminal amino acids of HemA may constitute a degradation tag, whose function is conditional and modified by the remainder of the protein in a heme-dependent way. Several models are discussed to explain why the turnover of HemA is promoted by Lon-ClpAP proteolysis only when sufficient heme is available.

ATP-Dependent Proteases↗
PubMed2000

In vitro hyperprocessing of Drosophila tRNAs by the catalytic RNA of RNase P the cloverleaf structure of tRNA is not always stable?

We have previously reported that the catalytic RNA subunit of RNase P of Escherichia coli (M1 RNA) cleaves Drosophila initiator methionine tRNA (tRNA(Met)i) within the mature tRNA sequence to produce specific fragments. This cleavage was dependent on the occurrence of an altered conformation of the tRNA substrate. We call this further cleavage hyperprocessing. In the present paper, to search for another tRNA that can be hyperprocessed in vitro, we used total mature tRNAs from Drosophila as substrates for the in vitro M1 RNA reaction. We found that some tRNAs can be hyperprocessed by M1 RNA and that two such tRNAs are an alanine tRNA and a histidine tRNA. Using mutant substrates of these tRNAs, we also show that the hyperprocessing by M1 RNA is dependent on the occurrence of altered conformations of these tRNAs. The altered conformations were very similar to that of tRNA(Met)i. We show here that M1 RNA can be used as a powerful tool to detect the alternative conformation of tRNAs. The relationship between these hyperprocessing reactions and stability of the tRNA structure will also be discussed.

Alanine↗
PubMed2003

Thermodynamic characterization of the binding of nucleotides to glycyl-tRNA synthetase.

The interaction of adenine nucleotides with glycyl-tRNA synthetase was examined by several experimental approaches. ATP and nonsubstrate ATP analogues render glycyl-tRNA synthetase more resistant to digestion by a number of proteases (thrombin, Arg-C, and chymotrypsin) at concentrations that correlate with their Michaelis constants or inhibition constants, consistent with their exerting an effect by binding at the ATP site. Glycine had little effect alone but potentiated the effect of ATP in increasing the resistance to thrombin digestion, consistent with the formation of an enzyme-bound adenylate. No protection from thrombin digestion was afforded by tRNA(gly). Binding constants were determined by isothermal titration calorimetry at 25 degrees C for ATP (2.5 x 10(5) M(-1)), AMPPNP (3.7 x 10(5) M(-1)), and AMPPCP (2.2 x 10(6) M(-1)). The nucleotides had similar values for DeltaH (-71 kJ mol(-1)), with values for TDeltaS that accounted for the differences in the binding constants. Near-ultraviolet CD spectra of the enzyme-nucleotide complexes indicate that the nucleotides are bound in the anti configuration. A glycyl-adenylate analogue, glycine sulfamoyl adenosine (GSAd), bound with a large value for DeltaH (-187 kJ mol(-1)), which was balanced by a large TDeltaS term to give a binding constant (3.7 x 10(6) M(-1)) only slightly larger than that of AMPPCP. Glycine binding to the enzyme could not be detected calorimetrically, and its presence did not change the thermodynamic parameters for binding of AMPPCP. AMPPNP and AMPPCP were not substrates for glycyl-tRNA synthetase. Analysis of the temperature dependence of ATP binding indicated that the heat capacity change is small, whereas the binding of GSAd is accompanied by a large negative heat capacity change (-2.6 kJ K(-1) mol(-1)). Titrations performed in buffers with different ionization enthalpies indicate that the large value for DeltaH for the adenylate analogue does not arise from a coupled protonation event. Differential scanning calorimetry indicated that glycyl-tRNA synthetase is stabilized by nucleotides. Unfolding of the protein is irreversible, and thermodynamic parameters for unfolding could therefore not be determined. The results are consistent with a significant conformational transition in glycyl-tRNA synthetase coupled to the binding of GSAd.

Adenosine Monophosphate↗
PubMed1975

Initiation of protein synthesis. Binding of messenger RNA.

Complexes between 30 S ribosomal subunits and fMet-tRNA are formed during incubation of 30 S subunits with fMet-tRNA and all other components for initiation of protein synthesis, except R17 bacteriophage RNA. That these complexes serve as intermediates in the binding of messenger RNA is demonstrated directly by the finding that upon addition of R17 RNA, fMet-tRNA in preformed fMet-RNA-30 S complexes preferentially enters fMet-tRNA-30 S-R17 RNA complexes. On the other hand, incubation of 30 S ribosomal subunits with R17 RNA and all other components for initiation except fMet-tRNA does not yield 30 S-R17 RNA complexes that can act subsequently as functional intermediates in the binding of fMet-tRNA: formation of fMet-tRNA-30 S-R17 RNA complexes does not occur when fMet-tRNA is added and further binding of R17 RNA to 30 S subunits is prevented by specific inhibitors. These experiments lead to an unambiguous order of events in the sequence of initiation, in which binding of fMet-tRNA to the small ribosomal subunit must occur before messenger RNA can be bound and phased correctly. Complexes between fMet-tRNA and 60 S subunits are in rapid equilibrium with the free components, and have a half-life of less than 2 min at 37 degrees. This explains why such complexes are not detected in sucrose gradients, unless they are first fixed with glutaraldehyde. Attachment of R17 RNA, however, results in formation of an fMet-tRNA-30 S-R17 RNA complex that is stabilized greatly; fMet-tRNA in this complex exchanges only very slowly with free fMet-tRNA. Initiation factor IF-3 has two functions in initiation. The first is to direct the binding of messenger RNA to the 30 S-fMet-tRNA complex. This function is not needed when initiation complex formation occurs on ApUpG triplets, in which case the second function of IF-3 is detected, that of providing free 30 S subunits for initiation. The ability of IF-3 to bind directly to R17 RNA may be related to its requirement in messenger RNA recognition. However, since IF-3 exhibits a greater affinity for the 30 S subunit than for R17 RNA, it appears that the recognition function of IF-3 is expressed while IF-3 is associated with the 30 S subunit.

Bacterial Proteins↗
PubMed2000

Gene-specific trans-regulatory functions of magnesium for chloroplast mRNA stability in higher plants.

In higher plant chloroplasts the accumulation of plastid-encoded mRNAs during leaf maturation is regulated via gene-specific mRNA stabilization. The half-lives of chloroplast RNAs are specifically affected by magnesium ions. psbA mRNA (D1 protein of photosystem II), rbcL mRNA (large subunit of ribulose-1,5-bisphosphate carboxylase), 16 S rRNA, and tRNA(His) gain stability at specific magnesium concentrations in an in vitro degradation system from spinach chloroplasts. Each RNA exhibits a typical magnesium concentration-dependent stabilization profile. It shows a cooperative response of the stability-regulated psbA mRNA and a saturation curve for the other RNAs. The concentration of free Mg(2+) rises during chloroplast development within a range sufficient to mediate gene-specific mRNA stabilization in vivo as observed in vitro. We suggest that magnesium ions are a trans-acting factor mediating differential mRNA stability.

5' Untranslated Regions↗
PubMed1992

Chloroplast heteroplasmicity is stabilized by an amber-suppressor tryptophan tRNA(CUA).

Photosynthesis-deficient mutants of the green alga Chlamydomonas reinhardtii were previously shown to arise from nonsense mutations within the chloroplast rbcL gene, which encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Photosynthesis-competent revertants of these nonsense mutants have often been found to be stably heteroplasmic, giving rise to both mutant and revertant cells during mitotic or meiotic divisions under nonselective growth conditions. It was proposed that the stable heteroplasmicity might arise from a balanced polymorphism of suppressor and wild-type alleles of a tRNA gene maintained within the polyploid chloroplast genome. In the present study, we have focused on determining the molecular basis for the heteroplasmicity of one such revertant, named R13-3C, which was recovered from the 18-7G amber (UAG) mutant. Restriction-enzyme analysis and DNA sequencing showed that the amber mutation is still present in the rbcL gene of the revertant strain. In contrast, DNA sequencing of the suspected tRNA(Trp) gene of the revertant revealed a mutation that would change its CCA anticodon to amber-specific CUA. This mutation was found to be heteroplasmic, being present in only 70% of the tRNA(Trp) gene copies. Under nonselective conditions, the suppressor mutation was lost from cells that also lost the revertant phenotype. We conclude that stable heteroplasmicity can arise as a balanced polymorphism of organellar alleles. This observation suggests that additional tRNA suppressors may be identified due to their heteroplasmic nature within polyploid genomes.

Animals↗
PubMed1992

Effect of the higher-order structure of tRNAs on the stability of hybrids with oligodeoxyribonucleotides: separation of tRNA by an efficient solution hybridization.

In the course of developing a method to purify a single tRNA species efficiently, we have examined hybridization efficiencies between some tRNAs and short oligodeoxyribonucleotide probes both by the filter and solution hybridization methods without denaturants. The hybridization efficiencies varied considerably among probes which are complementary to different regions of the tRNAs, although there was little efficiency variation in the probes toward DNA substrates including the same nucleotide sequence. This efficiency variation was shown to be due to tRNA-specific higher-order structures as well as a hypermodified nucleotide in the anticodon loop. Characterization of the tRNA-probe hybrids by both nondenaturing gel electrophoresis and chemical modification showed the existence of two stable hybridizing states as a function of ionic strength. Our results indicate that RNA molecules with a number of intramolecular base pairings are able to form stable hybrids with complementary sequences under nondenaturing conditions. On the basis of these data, an appropriate probe was designed to successfully purify yeast tRNA(Phe) by making a tRNA(Phe)-probe hybrid, which has a longer retention time in hydroxyapatite high performance liquid chromatography than the tRNA(Phe) itself.

Animals↗
PubMed1996

Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA.

Two residues of Escherichia coli elongation factor Tu involved in binding of aminoacyl-tRNA were identified and subjected to mutational analysis. Lys-89 and Asn-90 were each replaced by either Ala or Glu. The four single mutants were denoted K89A, K89E, N90A, and N90E, respectively. The mutants were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl-tRNA, which suggested an important role of Lys-89 and Asn-90 in tRNA binding. Furthermore, our results indicate helix B to be an important target site for nucleotide exchange factor EF-Ts. Also the mutants His-66 to Ala and His-118 to either Ala or Glu were characterized in an in vitro translation assay. Their functional roles are discussed in relation to the structure of elongation factor Tu in complex with aminoacyl-tRNA.

Amino Acid Sequence↗
PubMed2001

Reintroduction of a characterized Mit tRNA glycine mutation into yeast mitochondria provides a new tool for the study of human neurodegenerative diseases.

We report the identification and characterization of a new mutation (ts9) in the Saccharomyces cerevisiae mitochondrial genome, which was first genetically mapped in the tRNAgly region and further identified by means of sequencing as consisting of a G to A transition at position 30 in the tRNA. The mutation causes an almost complete disappearance of mature tRNAgly, while a second mitochondrial mutation with a compensatory C to T change restores it in normal quantities; this points to the importance of the strong bond between bases 30 and 40 of the anticodon stem in the stabilization of the tRNA. In addition to resulting in a clear-cut heat-sensitive phenotype, the ts9 mutation creates a new EcoRV restriction site. Both properties were used as markers to monitor the successful (re) introduction of the mutated allele into a wild-type mitochondrial genome through biolistic transformation. The mutant frequency in the progeny as well as the correct integration of the mutated allele at its proper site demonstrate the feasibility of this method for creating and investigating specific mitochondrial tRNA mutations. The method will provide important applications for the use of yeast as a model system of human mitochondrial pathologies.

Bacterial Proteins↗
PubMed2000

The tRNA-dependent activation of arginine by arginyl-tRNA synthetase requires inter-domain communication.

The tRNA-dependent amino acid activation catalyzed by mammalian arginyl-tRNA synthetase has been characterized. A conditional lethal mutant of Chinese hamster ovary cells that exhibits reduced arginyl-tRNA synthetase activity (Arg-1), and two of its derived revertants (Arg-1R4 and Arg-1R5) were analyzed at the structural and functional levels. A single nucleotide change, resulting in a Cys to Tyr substitution at position 599 of arginyl-tRNA synthetase, is responsible for the defective phenotype of the thermosensitive and arginine hyper-auxotroph Arg-1 cell line. The two revertants have a single additional mutation resulting in a Met222 to Ile change for Arg-1R4 or a Tyr506 to Ser change for Arg-1R5. The corresponding mutant enzymes were expressed in yeast and purified. The Cys599 to Tyr mutation affects both the thermal stability of arginyl-tRNA synthetase and the kinetic parameters for arginine in the ATP-PP(i) exchange and tRNA aminoacylation reactions. This mutation is located underneath the floor of the Rossmann fold catalytic domain characteristic of class 1 aminoacyl-tRNA synthetases, near the end of a long helix belonging to the alpha-helix bundle C-terminal domain distinctive of class 1a synthetases. For the Met222 to Ile revertant, there is very little effect of the mutation on the interaction of arginyl-tRNA synthetase with either of its substrates. However, this mutation increases the thermal stability of arginyl-tRNA synthetase, thereby leading to reversion of the thermosensitive phenotype by increasing the steady-state level of the enzyme in vivo. In contrast, for the Arg-1R5 cell line, reversion of the phenotype is due to an increased catalytic efficiency of the C599Y/Y506S double mutant as compared to the initial C599Y enzyme. In light of the location of the mutations in the 3D structure of the enzyme modeled using the crystal structure of the closely related yeast arginyl-tRNA synthetase, the kinetic analysis of these mutants suggests that the obligatory tRNA-induced activation of the catalytic site of arginyl-tRNA synthetase involves interdomain signal transduction via the long helices that build the tRNA-binding domain of the enzyme and link the site of interaction of the anticodon domain of tRNA to the floor of the active site.

Acylation↗
PubMed1979

The effect of amino acids on the temperature sensitive phenotype of the mammalian leucyl-tRNA synthetase mutant tsHl and its revertants.

The temperature sensitive leucyl-tRNA synthetase mutant tsHl and two revertants have been compared to the parental Chinese hamster ovary cells with respect to the effects of amino acid concentrations in the medium on growth. Elevating the leucine concentration 30- or 100-fold allowed tsHl to grow exponentially at 38.5 degrees C, normally the nonpermissive temperature. Partial revertants that had recovered some enzyme activity required smaller supplements for growth. Measurements of the leucine pools indicated that they respond directly to the extracellular leucine concentration and may mediate the effect. Use of combinations of amino acids confirmed that isoleucine has a similar though weaker effect on tsHl and identified an even weaker protection by valine. The triple combination of leucine, isoleucine and valine was a much more efficient medium supplement and three times normal concentrations of these amino acids supported growth of tsHl at 38.5 degrees C. It is postulated that they are acting at their respective aminoacyl-tRNA synthetases to help stabilize a complex which also contains the mutant leucyl-tRNA synthetase. The pool size measurements also showed that the leucine pools of tsHl and a revertant increased 2-fold more in a response to increased temperature than those of WT. It is suggested that this is a regulatory response to low leucyl-tRNA synthetase activity and is important in determining growth phenotypes.

Amino Acyl-tRNA Synthetases↗
PubMed2006

Structure of the 70S ribosome complexed with mRNA and tRNA.

The crystal structure of the bacterial 70S ribosome refined to 2.8 angstrom resolution reveals atomic details of its interactions with messenger RNA (mRNA) and transfer RNA (tRNA). A metal ion stabilizes a kink in the mRNA that demarcates the boundary between A and P sites, which is potentially important to prevent slippage of mRNA. Metal ions also stabilize the intersubunit interface. The interactions of E-site tRNA with the 50S subunit have both similarities and differences compared to those in the archaeal ribosome. The structure also rationalizes much biochemical and genetic data on translation.

Anticodon↗
PubMedDate not supplied

tRNA-ribosome interactions.

Direct measurements of the rates of dissociation of dipeptidyl-tRNA from the ribosome show that hyperaccurate SmP and SmD ribosomes have unstable A-site binding of peptidyl-tRNA, while P-site binding is extremely stable in relation to the wild type. Error-prone Ram ribosomes, on the other hand, have stable A-site and unstable P-site binding of peptidyl-tRNA. At least for these mutant ribosomes, we conclude that stabilization of peptidyl-tRNA in one site destabilizes binding in the other. Elongation factor Tu (EF-Tu) undergoes a dramatic structural transition from its GDP-bound form to its active GTP-bound form, in which it binds aa-tRNA (aminoacyl-tRNA) in ternary complex. The effects of substitution mutations at three sites in domain I of EF-Tu, Gln124, Leu120, and Tyr160, all of which point into the domain I-domain III interface in both the GTP and GDP conformations of EF-Tu, were examined. Mutations at each position cause large reductions in aa-tRNA binding. An attractive possibility is that the mutations alter the domain I-domain III interface such that the switching of EF-Tu between different conformations is altered, decreasing the probability of aa-tRNA binding. We have previously found that two GTPs are hydrolyzed per peptide bond on EF-Tu, the implication being that two molecules of EF-Tu may interact on the ribosome to catalyze the binding of a single aa-tRNA to the A-site. More recently we found that ribosomes programmed with mRNA constructs other than poly(U), including the sequence AUGUUUACG, invariably use two GTPs per peptide bond in EF-Tu function. Other experiments measuring the protection of aa-tRNA from deacylation or from RNAse A attack show that protection requires two molecules of EF-Tu, suggesting an extended ternary complex. To remove remaining ambiguities in the interpretion of these experiments, we are making direct molecular weight determinations with neutron scattering and sedimentation-diffusion techniques.

Bacterial Proteins↗