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At least 235 records · Page 13Linked to original sources
PubMed1993

Use of binding energy in catalysis: optimization of rate in a multistep reaction.

The role of binding energy in optimizing the overall rate of catalysis by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus has been investigated by measuring the rate constants for transfer of tyrosine from engineered mutants to tRNA. The residues chosen for mutation are those that were previously identified as binding tyrosyl adenylate and contributing to the rate constant for its formation. It was previously found that tighter binding of the tyrosyl adenylate was accompanied by an increase in the rate constant for its formation. A new linear free energy relationship is presented that links the two. We now find that the rate constant for transfer of Tyr from Tyr-AMP to tRNA decreases with increasing stability of the E.Tyr-AMP complex on mutation of Thr-51. Position 51 is the one that is found to be the most variable of the binding-site residues among the enzymes from different species. The tightness of binding of the intermediate is thus a compromise, since stabilizing the intermediate speeds up the first step but slows down the second. The rate constants for activation and transfer by wild-type enzymes are very similar, which is optimal for the rate of the overall reaction. Those for the mutants diverge so that the rate of overall catalysis is lower.

Adenosine Monophosphate↗
PubMed2000

Human immunodeficiency virus type-1 reverse transcription can be inhibited in vitro by oligonucleotides that target both natural and synthetic tRNA primers.

Reverse transcription of human immunodeficiency virus type-1 is primed by cellular tRNA(Lys3), which is selectively packaged into viral particles where it is bound at its 3' terminus to a complementary sequence of viral RNA termed the primer binding site (PBS). Since cellular tRNA(Lys3) is highly conserved, it might conceivably serve as a good target for novel antagonists to block reverse transcriptase (RT) activity. In this study, we have examined a number of antisense oligodeoxyribonucleotides (ODNs) that are complementary to different parts of the tRNA primer and, therefore, may interfere with the initiation of RT-mediated DNA synthesis. We found that the stability of complexes between synthetic tRNA(Lys3 )and ODNs was significantly increased when binding occurred via sequences involved in tertiary interactions of the tRNA. In particular, ODNs with complementarity to both the variable and TPsiC stem-loop of tRNA(Lys3 )bound with high affinity to both free tRNA(Lys3 )as well as to the binary tRNA(Lys3)/RNA complex. As a result, the initiation of DNA synthesis was severely compromised under these conditions. Moreover, RT-associated RNase H activity recognized the tRNA within this ternary tRNA(Lys3)/RNA/ODN complex as an RNA template and initiated its degradation. Both this RNase H degradation of tRNA(Lys3 )as well as the altered structure of the tRNA/RNA complex, due to the binding of the ODN, contributed to the inhibition of synthesis of viral DNA. The initiation of RT activity was almost completely blocked when using ODNs that interfered with intermolecular tRNA/RNA interactions that involved both the PBS and sequences outside the PBS. Similar findings were obtained with natural preparation of tRNA(Lys3).

Base Sequence↗
PubMed1983

Isolation of Chinese hamster ovary cell mutants defective in the regulation of leucine transport.

We have isolated mutants defective in the regulation of leucine transport by selecting temperature-resistant revertants from the CHO-tsH1 strain, a temperature-sensitive leucyl-tRNA synthetase mutant of the Chinese hamster ovary cell line. In each revertant, there is a stable 2- to 3-fold enhancement in the activity of transport System L, which serves for the uptake of branched-chain amino acids. This increased transport is reflected by an increase in the Vmax for System L transport without a significant change in the Km value and results in increased intracellular pools of leucine. The thermal stability of the leucyl-tRNA synthetase activity in each of these revertants is not changed significantly from that of the starting strain, CHO-tsH1. We conclude from these studies that the temperature resistance in the revertants arises from alterations in the regulation of transport System L, leading to constitutively high System L transport and increased intracellular pools of leucine, complementing the leucyl-tRNA synthetase defect.

Amino Acyl-tRNA Synthetases↗
PubMed1992

Codon-anticodon pairing. A model for interacting codon-anticodon duplexes located at the ribosomal A- and P-sites.

The interaction between two codon-anticodon duplexes of the ribosomal A- and P-site-bound tRNAs is the key feature of the proposed model. This interaction prohibits non-canonical base pairing at the first and second positions of the codon and controls base pairing at the third position (wobbling rules ensuing from the model are in good accord with those generated from experiments). The model is capable of predicting codon context effects. It follows from the model that modifications of the first anticodon residue of the P-site tRNA can affect the stability of the A-site duplex, and that the translation of a DNA single chain analogue of mRNA should be accompanied by non-canonical base pairing at all three positions of the codon. These predictions of the model can be subjected to experimental tests.

Anticodon↗
PubMed2005

Splitting of the posttermination ribosome into subunits by the concerted action of RRF and EF-G.

After peptide release by a class-1 release factor, the ribosomal subunits must be recycled back to initiation. We have demonstrated that the distance between a strong Shine-Dalgarno (SD) sequence and a codon in the P site is crucial for the binding stability of the deacylated tRNA in the P site of the posttermination ribosome and the in-frame maintenance of its mRNA. We show that the elongation factor EF-G and the ribosomal recycling factor RRF split the ribosome into subunits in the absence of initiation factor 3 (IF3) by a mechanism that requires both GTP and GTP hydrolysis. Taking into account that EF-G in the GTP form and RRF bind with positive cooperativity to the free 50S subunit but with negative cooperativity to the 70S ribosome, we suggest a mechanism for ribosome recycling that specifies distinct roles for EF-G, RRF, and IF3.

Base Sequence↗
PubMed1999

Chloroplast ribonucleoproteins are associated with both mRNAs and intron-containing precursor tRNAs.

Tobacco chloroplasts possess five conserved ribonucleoproteins (cpRNPs). To elucidate the function of cpRNPs we analyzed their localization and target nucleic acid molecules in chloroplasts. Immunoprecipitation of the stromal extract and Northern analysis revealed that cpRNPs are associated in vivo with not only various species of chloroplast mRNAs but also intron-containing precursor (pre-) tRNAs. This observation strongly suggests that cpRNPs are involved in RNA processing, including mRNA stability and pre-tRNA splicing.

Blotting, Northern↗
PubMed1988

Physical mapping of large DNA by chromosome fragmentation.

A technique is described for physically positioning any cloned DNA on a native or artificial Saccharomyces cerevisiae chromosome. The technique involves splitting a chromosome at a specific site by transformation with short linear molecules containing the cloned DNA at one end and telomeric sequences at the other. Recombination between the end of the linear molecules and homologous chromosomal sequences gives rise to chromosome fragments comprising all sequences distal or proximal to the mapping site depending on the orientation of the cloned DNA. The recombinant products are recovered by screening for stabilization of a suppressor tRNA on the linear molecules using a colony color assay. The cloned DNA is positioned relative to the chromosome ends by sizing the chromosomal fragments using alternating contour-clamped homogeneous electric field gel electrophoresis. Application of this technique to organisms other than S. cerevisiae and to the analysis of exogenous DNA cloned in yeast is discussed.

Chromosome Mapping↗
PubMed1997

Identification of functional conserved residues of CTP:glycerol-3-phosphate cytidylyltransferase. Role of histidines in the conserved HXGH in catalysis.

The CTP:glycerol-3-phosphate cytidylyltransferase (GCT) of Bacillus subtilis has been shown to be similar in primary structure to the CTP:phosphocholine cytidylyltransferases of several organisms. To identify the residues of this cytidylyltransferase family that function in catalysis, the conserved hydrophilic amino acid residues plus a conserved tryptophan of the GCT were mutated to alanine. The most dramatic losses in activity occurred with H14A and H17A; these histidine residues are part of an HXGH sequence similar to that found in class I aminoacyl-tRNA synthetases. The kcat values for H14A and H17A were decreased by factors of 5 x 10(-5) and 4 x 10(-4), respectively, with no significant change in Km values. Asp-11, which is found near the HXGH sequence in the cytidylyltransferases but not aminoacyl-tRNA synthetases, was also important for activity, with the D11A mutation decreasing activity by a factor of 2 x 10(-3). Several residues found in the sequence RTEGISTT, a signature sequence for this cytidylyltransferase family, as well as other isolated residues were also shown to be important for activity, with kcat values decreasing by factors of 0.14-4 x 10(-4). The Km values of three mutant enzymes, D38A, W74A, and D94A, for both CTP and glycerol-3-phosphate were 6-130-fold higher than that of the wild-type enzyme. Mutant enzymes were analyzed by two-dimensional NMR to determine if the overall structures of the enzymes were intact. One of the mutant enzymes, D66A, was defective in overall structure, but several of the others, including H14A and H17A, were not. These results indicate that His-14 and His-17 play a role in catalysis and suggest that their role is similar to the role of the His residues in the HXGH sequence in class I aminoacyl-tRNA synthetases, i.e. to stabilize a pentacoordinate transition state.

Amino Acid Sequence↗
PubMed1998

Characterization of cDNAs encoding the p44 and p35 subunits of human translation initiation factor eIF3.

Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit complex that plays a central role in the initiation of translation. It binds to 40 S ribosomal subunits resulting in dissociation of 80 S ribosomes, stabilizes initiator methionyl-tRNA binding to 40 S subunits, and is required for mRNA binding. eIF3 has an aggregate molecular mass of approximately 600 kDa and comprises at least 10 subunits. The cDNAs encoding eight of the subunits have been cloned previously (p170, p116, p110, p66, p48, p47, p40, and p36). Here we report the cloning and characterization of human cDNAs encoding two more subunits of human eIF3, namely eIF3-p44 and eIF3-p35. These proteins are immunoprecipitated by affinity-purified anti-eIF3-p170 antibodies, indicating they are components of the eIF3 complex. Far Western analysis shows that eIF3-p44 interacts strongly and specifically with the eIF3-p170 subunit, and weakly with p116/p110, p66, p40, and itself. eIF3-p44 contains an RNA recognition motif near its C terminus. Northwestern blotting shows that eIF3-p44 binds 18 S rRNA and beta-globin mRNA. Possession of cloned cDNAs encoding all 10 subunits of eIF3 provides the tools necessary to elucidate the functions of the individual subunits and the structure of the eIF3 complex.

Amino Acid Sequence↗
PubMed1999

Two yeast La motif-containing proteins are RNA-binding proteins that associate with polyribosomes.

We have characterized two Saccharomyces cerevisiae proteins, Sro9p and Slf1p, which contain a highly conserved motif found in all known La proteins. Originally described as an autoantigen in patients with rheumatic disease, the La protein binds to newly synthesized RNA polymerase III transcripts. In yeast, the La protein homologue Lhp1p is required for the normal pathway of tRNA maturation and also stabilizes newly synthesized U6 RNA. We show that deletions in both SRO9 and SLF1 are not synthetically lethal with a deletion in LHP1, indicating that the three proteins do not function in a single essential process. Indirect immunofluorescence microscopy reveals that although Lhp1p is primarily localized to the nucleus, Sro9p is cytoplasmic. We demonstrate that Sro9p and Slf1p are RNA-binding proteins that associate preferentially with translating ribosomes. Consistent with a role in translation, strains lacking either Sro9p or Slf1p are less sensitive than wild-type strains to certain protein synthesis inhibitors. Thus, Sro9p and Slf1p define a new and possibly evolutionarily conserved class of La motif-containing proteins that may function in the cytoplasm to modulate mRNA translation.

Amino Acid Sequence↗
PubMed1995

Transition state stabilization by the 'high' motif of class I aminoacyl-tRNA synthetases: the case of Escherichia coli methionyl-tRNA synthetase.

Methionyl-tRNA synthetase belongs to the class I aminoacyl-tRNA synthetase family characterized both by a catalytic center built around a Rossmann Fold and by the presence of the two peptidic marker sequences HIGH and KMSKS. In this study, the role of the 21HLGH24 motif of Escherichia coli methionyl-tRNA synthetase was studied in a systematic fashion by site-directed mutagenesis. It is shown that the two histidine residues play a crucial role in the catalysis of the methionyl adenylate formation by participating in the stabilisation of the ATP phosphate chain during the transition state. Moreover, the results suggest the involvement of the epsilon-imino group of histidine 21 and of the delta-imino group of histidine 24. Notably, the substitution of either the leucine or the glycine residue of the HLGH motif by alanine had no effect on the catalysis. From the data and from other studies with class I aminoacyl-tRNA synthetases, concomitant positive contributions of the HIGH and KMSKS sequences to reach the transition state of aminoacyl adenylate formation can be envisaged.

Adenosine Monophosphate↗
PubMedDate not supplied

From DNA sequence to application: possibilities and complications.

The development of sophisticated genetic tools during the past 15 years have facilitated a tremendous increase of fundamental and application-oriented knowledge of lactic acid bacteria (LAB) and their bacteriophages. This knowledge relates both to the assignments of open reading frames (ORF's) and the function of non-coding DNA sequences. Comparison of the complete nucleotide sequences of several LAB bacteriophages has revealed that their chromosomes have a fixed, modular structure, each module having a set of genes involved in a specific phase of the bacteriophage life cycle. LAB bacteriophage genes and DNA sequences have been used for the construction of temperature-inducible gene expression systems, gene-integration systems, and bacteriophage defence systems. The function of several LAB open reading frames and transcriptional units have been identified and characterized in detail. Many of these could find practical applications, such as induced lysis of LAB to enhance cheese ripening and re-routing of carbon fluxes for the production of a specific amino acid enantiomer. More knowledge has also become available concerning the function and structure of non-coding DNA positioned at or in the vicinity of promoters. In several cases the mRNA produced from this DNA contains a transcriptional terminator-antiterminator pair, in which the antiterminator can be stabilized either by uncharged tRNA or by interaction with a regulatory protein, thus preventing formation of the terminator so that mRNA elongation can proceed. Evidence has accumulated showing that also in LAB carbon catabolite repression in LAB is mediated by specific DNA elements in the vicinity of promoters governing the transcription of catabolic operons. Although some biological barriers have yet to be solved, the vast body of scientific information presently available allows the construction of tailor-made genetically modified LAB. Today, it appears that societal constraints rather than biological hurdles impede the use of genetically modified LAB.

Bacteriophages↗
PubMedDate not supplied

[Minor secondary-structure variation in the 5'-untranslated region of the beta-globin mRNA changes the concentration requirements for eIF2].

Nucleotide sequence changes increasing the number of paired bases without producing stable secondary structure elements in the 5'-untranslated region (5'-UTR) of the beta-globin mRNA had a slight effect on its translation in rabbit reticulocyte lysate at its low concentration and dramatically decreased translation efficiency at a high concentration. The removal of paired regions restored translation. Addition of purified eIF2 to the lysate resulted in equal translation efficiencies of templates differing in structure of 5'-UTR. A similar effect was observed for p50, a major mRNP protein. Other mRNA-binding initiation factors, eIF4F and eIF3B, had no effect on the dependence of translation efficiency on mRNA concentration. Analysis of the assembly of the 48S initiation complex from its purified components showed that less eIF2 is required for translation initiation on the beta-globin mRNA than on its derivative containing minor secondary structure elements in 5'-UTR. According to a model proposed, eIF2 not only delivers Met-tRNA, but it also stabilizes the complex of the 40S ribosome subunit with 5'-UTR, which is of particular importance for translation initiation on templates with structured 5'-UTR.

5' Untranslated Regions↗
PubMed1998

Solution structure of a substrate for the archaeal pre-tRNA splicing endonucleases: the bulge-helix-bulge motif.

The structure of the bulge-helix-bulge motif that constitutes the intron/exon splice site in H. volcanii pre-tRNATrp has been determined by NMR spectroscopy. The conformations of the two 3 nt bulges, where the pre-tRNA is cleaved, are stabilized by stacking interactions between bulge nucleotides and bases in the adjacent Watson-Crick helices and by a network of backbone hydrogen bonds. Both bulges are presented on the same minor groove face of the central 4 bp helix, and the overall structure has approximate two-fold symmetry, which makes it well-suited for attack by archaeal splicing endonucleases, which are symmetric dimers.

Archaeal Proteins↗
PubMedDate not supplied

[The analysis of monomer sequences in protein and tRNA and the manifestation of the compensation of pathogenic deviations in their evolution].

The sequence analysis of proteins and mitochondrial tRNA indicated that substitutions inducing the pathogenesis in humans often occur in normal individuals of other species having no signs of pathology. The analysis of the structural stability of the protein and tRNA molecules showed that the harmful effect of pathogenic substitutions is often neutralized by other compensatory substitutions, which restore the required normal stability of the structure. A further study of this phenomenon will probably lead to new methods of treatment of genetic pathology, which would be based not on the correction of one substitution but on the correction of the stability of the molecule as an entire functional unit.

Animals↗
PubMed2000

New chromatographic and biochemical strategies for quick preparative isolation of tRNA.

A combination of hydrophobic chromatography on phenyl-Sepharose and reversed phase HPLC was used to purify individual tRNAs with high specific activity. The efficiency of chromatographic separation was enhanced by biochemical manipulations of the tRNA molecule, such as aminoacylation, formylation of the aminoacyl moiety and enzymatic deacylation. Optimal combinations are presented for three different cases. (i) tRNA(Phe) from Escherichia coli. This species was isolated by a combination of low pressure phenyl-Sepharose hydrophobic chromatography with RP-HPLC. (ii) tRNA(Ile) from E. coli: Aminoacylation increases the retention time for this tRNA in RP-HPLC. The recovered acylated intermediate is deacylated by reversion of the aminoacylation reaction and submitted to a second RP-HPLC run, in which deacylated tRNA(Ile) is recovered with high specific activity. (iii) tRNA(i)(Met) from Saccharomyces cerevisiae. The aminoacylated form of this tRNA is unstable. To increase stability, the aminoacylated form was formylated using E.coli: enzymes and, after one RP-HPLC step, the formylated derivative was deacylated using peptidyl-tRNA hydrolase from E.COLI: The tRNA(i)(Met) recovered after a second RP-HPLC run exhibited electrophoretic homogeneity and high specific activity upon aminoacylation. These combinations of chromatographic separation and biochemical modification can be readily adapted to the large-scale isolation of any particular tRNA.

Acylation↗
PubMed1975

Studies on the binding of acylaminoacyl-tRNA to rat liver 60S ribosomal subunits and its participation in the peptidyltransferase reaction.

Peptidyltransferase with rat liver 60S subunits can be measured by the reaction between exogenous acylaminoacyl-tRNA and puromycin to form acylaminoacylpuromycin in the presence of 33% methanol, 0.3 M KCl, and 4 mM MgCl2. An assay system has been developed that allows examination of the binding of acetylphenylalanyl-tRNA to the ribosomal subunit "P" site, the transpeptidation of the 60S-bound substrate to puromycin, and the requirements for these individual steps. Binding of acetylphenylalanyl-tRNA to 60S subunits is stimulated several-fold by the addition of methanol, but the extent of binding in alcohol is the same in 60 as in 300 mM KCl containing solutions. Formation of acetylphenylalanyl-puromycin from 60S-acetylphenylalanyl-tRNA complex and puromycin stringently requires alcohol and the initial rate of the reaction is markedly greater at 300 mM KCl than at 60 mM KCl concentrations. Thus, alcohol and high concentrations of monovalent cation affect the reaction of an event subsequent to the binding of substrate to the "P" site. Preincubation of 60S subunits with poly(U), which stimulates the overall peptidyltransferase reaction, does not affect the amount of acetylphenylalanyl-tRNA that is bound to the particles; however, it markedly stimulates the initial rate of the transpeptidation reaction between 60S-acetylphenylalanyl-tRNA complex and puromycin. The codon specificity and the failure to affect binding with poly(U) suggest a role for the polynucleotide in the alignment or stabilization of the acylaminoacyl-tRNA on the "P" site rather than an effect on binding to either of the two particle sites or on the peptidyltransferase "active center." The effect of 40S subunits, which inhibit the overall peptidyltransferase reaction, on the binding of substrate could not be clearly interpreted since all three preparations, 60S subunits, 40S subunits, and combinations of 60S plus 40S particles, appear to bind acetylphenylalanyl-tRNA in the presence of methanol. However, the initial rate of peptide bond formation is several times greater with 60S-acetylphenylalanyl-tRNA complex than with 60S plus 40S particles containing bound acetylphenylalanyl-tRNA and the addition of 40S subunits to preformed 60S-acetylphenylalanyl-tRNA complex during the transpeptidation phase of the reaction in methanol does not affect the rate of peptide bond formation. Thus, 40S subunits seem to inhibit peptidyltransferase by forming less reactive particles in aqueous solutions. Contd.

Acyltransferases↗