Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “tRNA stability”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 721 records · Page 40Linked to original sources
PubMed2000

Intact aminoacyl-tRNA is required to trigger GTP hydrolysis by elongation factor Tu on the ribosome.

GTP hydrolysis by elongation factor Tu (EF-Tu) on the ribosome is induced by codon recognition. The mechanism by which a signal is transmitted from the site of codon-anticodon interaction in the decoding center of the 30S ribosomal subunit to the site of EF-Tu binding on the 50S subunit is not known. Here we examine the role of the tRNA in this process. We have used two RNA fragments, one which contains the anticodon and D hairpin domains (ACD oligomer) derived from tRNA(Phe) and the second which comprises the acceptor stem and T hairpin domains derived from tRNA(Ala) (AST oligomer) that aminoacylates with alanine and forms a ternary complex with EF-Tu. GTP. While the ACD oligomer and the ternary complex containing the Ala-AST oligomer interact with the 30S and 50S A site, respectively, no rapid GTP hydrolysis was observed when both were bound simultaneously. The presence of paromomycin, an aminoglycoside antibiotic that binds to the decoding site and stabilizes codon-anticodon interaction in unfavorable coding situations, did not increase the rate of GTP hydrolysis. These results suggest that codon recognition as such is not sufficient for GTPase activation and that an intact tRNA molecule is required for transmitting the signal created by codon recognition to EF-Tu.

Anticodon↗
PubMed1984

Structure of intron-containing tRNA precursors. Analysis of solution conformation using chemical and enzymatic probes.

Using chemical and enzymatic structure-specific probes adapted to rapid gel sequencing techniques, we have analyzed the solution conformations of precursors to two yeast tRNAs which contain an intervening sequence, pre-tRNAPhe and pre-tRNATyr. Interpretation of the data was greatly facilitated by performing direct mature/precursor tRNA comparisons. In addition, the effects of tertiary interactions on probe specificity could be evaluated from the results obtained with mature tRNAPhe, whose crystal structure is known. We find: 1) the folding of the precursor CCA terminus, acceptor stem, T psi C stem, variable loop, anticodon stem, and D stem identical with that of the equivalent regions in the cognate, mature tRNA. 2) The T psi C loop and D loop appear to vary slightly in tertiary structure between mature and precursor species. 3) The precursors contain a helix involving the anticodon triplet and a complementary sequence in the intron. 4) The stability of this helix is much greater for pre-tRNAPhe than for pre-tRNATyr. 5) The splice sites for both precursors are located in single-stranded loops. These results bear out predictions based on genetic analyses and are consistent with the view that recognition of universally conserved features of tRNA structure allows all tRNA precursors containing intervening sequences to be processed by a single splicing apparatus.

Aldehydes↗
PubMed2000

Importance of discriminator base stacking interactions: molecular dynamics analysis of A73 microhelix(Ala) variants.

Transfer of alanine from Escherichia coli alanyl-tRNA synthetase (AlaRS) to RNA minihelices that mimic the amino acid acceptor stem of tRNA(Ala) has been shown, by analysis of variant minihelix aminoacylation activities, to involve a transition state sensitive to changes in the 'discriminator' base at position 73. Solution NMR has indicated that this single-stranded nucleotide is predominantly stacked onto G1 of the first base pair of the alanine acceptor stem helix. We report the activity of a new variant with the adenine at position 73 substituted by its non-polar isostere 4-methylindole (M). Despite lacking N7, this analog is well tolerated by AlaRS. Molecular dynamics (MD) simulations show that the M substitution improves position 73 base stacking over G1, as measured by a stacking lifetime analysis. Additional MD simulations of wild-type microhelix(Ala) and six variants reveal a positive correlation between N73 base stacking propensity over G1 and aminoacylation activity. For the two DeltaN7 variants simulated we found that the propensity to stack over G1 was similar to the analogous variants that contain N7 and we conclude that the decrease in aminoacylation efficiency observed upon deletion of N7 is likely due to loss of a direct stabilizing interaction with the synthetase.

Acylation↗
PubMed1997

The yeast La protein is required for the 3' endonucleolytic cleavage that matures tRNA precursors.

Although the La autoantigen binds to the 3' ends of all nascent polymerase III transcripts, its function in vivo has long been unclear. Although S. cerevisiae cells lacking the La protein homolog Lhp1p are viable, cells containing a mutation that disrupts the anticodon stem of tRNA(Ser)CGA require Lhp1p for growth. We demonstrate that for the wild-type pre-tRNA(Ser)CGA and other pre-tRNAs, Lhp1p is required for the normal endonucleolytic removal of the 3' trailer sequence. In cells lacking Lhp1p, the 3' trailer is removed by exonuclease(s). Although maturation of the mutant pre-tRNA(Ser)CGA requires Lhp1p, introduction of a second mutation that restores base pairing eliminates the requirement. We propose that binding by Lhp1p stabilizes pre-tRNAs in conformations that allow the 3' endonucleolytic cleavage to occur.

Autoantigens↗
PubMed1999

The crystal structure of a novel bacterial adenylyltransferase reveals half of sites reactivity.

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in bacteria that catalyses a rate-limiting step in coenzyme A (CoA) biosynthesis, by transferring an adenylyl group from ATP to 4'-phosphopantetheine, yielding dephospho-CoA (dPCoA). Each phosphopantetheine adenylyltransferase (PPAT) subunit displays a dinucleotide-binding fold that is structurally similar to that in class I aminoacyl-tRNA synthetases. Superposition of bound adenylyl moieties from dPCoA in PPAT and ATP in aminoacyl-tRNA synthetases suggests nucleophilic attack by the 4'-phosphopantetheine on the alpha-phosphate of ATP. The proposed catalytic mechanism implicates transition state stabilization by PPAT without involving functional groups of the enzyme in a chemical sense in the reaction. The crystal structure of the enzyme from Escherichia coli in complex with dPCoA shows that binding at one site causes a vice-like movement of active site residues lining the active site surface. The mode of enzyme product formation is highly concerted, with only one trimer of the PPAT hexamer showing evidence of dPCoA binding. The homologous active site attachment of ATP and the structural distribution of predicted sequence-binding motifs in PPAT classify the enzyme as belonging to the nucleotidyltransferase superfamily.

Binding Sites↗
PubMed2000

Tertiary core rearrangements in a tight binding transfer RNA aptamer.

Guided by an in vitro selection experiment designed to obtain tight binding aptamers of Escherichia coli glutamine specific tRNA (tRNAGln) for glutaminyl-tRNA synthetase (GlnRS), we have engineered a tRNA mutant in which the five-nucleotide variable loop sequence 5'-44CAUUC48-3' is replaced by 5'-44AGGU48-3'. This mutant tRNA binds to GlnRS with 30-fold improved affinity compared to the wild type. The 2.7 A cocrystal structure of the RNA aptamer-GlnRS complex reveals major rearrangements in the central tertiary core of the tRNA, while maintaining an RNA-protein interface identical to the wild type. The repacked RNA core features a novel hydrogen bonding arrangement of the trans Levitt pair G15-U48, a new sulfate binding pocket in the major groove, and increased hydrophobic stacking interactions among the bases. These data suggest that enhanced protein binding to a mutant globular RNA can arise from stabilization of RNA tertiary interactions rather than optimization of RNA-protein contacts.

Amino Acyl-tRNA Synthetases↗
PubMed1987

Dissection of the structure and activity of the tyrosyl-tRNA synthetase by site-directed mutagenesis.

To understand an enzyme reaction, one has to characterize the bound substrates, intermediates, products, and transition states on the reaction pathway and determine the interaction energies between them and the enzyme as the reaction proceeds. Site-directed mutagenesis is invaluable in this task, enabling the systematic dissection of the active site. Residues involved in catalysis may be detected and the energetics probed. The contributions of each hydrogen-bonding site chain in the active site of the tyrosyl-tRNA synthetase to binding and catalysis are being determined by making sensible mutations, which remove defined interactions with the substrates. The difference in free energy between complexes of wild-type and mutant enzymes gives the apparent binding energy of the relevant side chain in each complex. By this means, the following have been determined: the contributions of different types of hydrogen bonds to specificity; their roles in catalysis; the importance of enzyme--substrate versus enzyme--transition-state versus enzyme--intermediate complementarity; the fine tuning of enzyme catalysis during "evolution"; and the existence of linear free energy relationships between rate and binding constants. Prior to these studies, the mechanism of activation by the aminoacyl-tRNA synthetases was totally unknown. It is now seen that catalysis results solely from the use of binding energy. There are residues that do not bind the substrates in the ground state but stabilize just the transition state, consistent with the classical ideas of Haldane and Pauling of enzyme--transition-state complementarity. There are, however, regions of the protein that bind the ribose ring more tightly in the intermediate tyrosyl adenylate than in the transition state.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acyl-tRNA Synthetases↗
PubMed2001

Function of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase in RNA splicing. Role of the idiosyncratic N-terminal extension and different modes of interaction with different group I introns.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by helping the intron RNA fold into the catalytically active structure. The regions required for splicing include an idiosyncratic N-terminal extension, the nucleotide-binding fold domain, and the C-terminal RNA-binding domain. Here, we show that the idiosyncratic N-terminal region is in fact comprised of two functionally distinct parts: an upstream region consisting predominantly of a predicted amphipathic alpha-helix (H0), which is absent from bacterial tyrosyl-tRNA synthetases (TyrRSs), and a downstream region, which contains predicted alpha-helices H1 and H2, corresponding to features in the X-ray crystal structure of the Bacillus stearothermophilus TyrRS. Bacterial genetic assays with libraries of CYT-18 mutants having random mutations in the N-terminal region identified functionally important amino acid residues and supported the predicted structures of the H0 and H1 alpha-helices. The function of N and C-terminal domains of CYT-18 was investigated by detailed biochemical analysis of deletion mutants. The results confirmed that the N-terminal extension is required only for splicing activity, but surprisingly, at least in the case of the N. crassa mitochondrial (mt) large ribosomal subunit (LSU) intron, it appears to act primarily by stabilizing the structure of another region that interacts directly with the intron RNA. The H1/H2 region is required for splicing activity and TyrRS activity with the N. crassa mt tRNA(Tyr), but not for TyrRS activity with Escherichia coli tRNA(Tyr), implying a somewhat different mode of recognition of the two tyrosyl-tRNAs. Finally, a CYT-18 mutant lacking the N-terminal H0 region is totally defective in binding or splicing the N. crassa ND1 intron, but retains substantial residual activity with the mt LSU intron, and conversely, a CYT-18 mutant lacking the C-terminal RNA-binding domain is totally defective in binding or splicing the mt LSU intron, but retains substantial residual activity with the ND1 intron. These findings lead to the surprising conclusion that CYT-18 promotes splicing via different sets of interactions with different group I introns. We suggest that these different modes of promoting splicing evolved from an initial interaction based on the recognition of conserved tRNA-like structural features of the group I intron catalytic core.

Adenosine Monophosphate↗
PubMed1989

Insertion of new sequences into the catalytic domain of an enzyme.

Activities of enzymes can be modified by the replacement of active-site amino acids with residues that strengthen specific interactions with substrates or that alter the specificity. The scope for engineered enzymes would be broadened if additional, new sequences could be inserted into a catalytic domain. Properly designed, these sequences could encode new ligand binding sites, be intermediates in the construction of chimeric enzymes, or alter the internal flexibility and "breathing" modes of the active-site region. As a first step toward this objective, we inserted oligopeptides of up to 14 amino acids into various locations within an 82 amino acid region of the adenylate synthesis domain of Escherichia coli methionyl-tRNA synthetase. These sites include ones that are flanked by sequences that are conserved between the proteins from E. coli and the yeast Saccharomyces cerevisiae and those that are essential for activity and stability. We found that all of the insertional mutants are stable and some have catalytic parameters for adenylate synthesis that are comparable to those of the wild-type enzyme. Thus, such an approach may provide for a variety of novel applications.

Amino Acid Sequence↗
PubMed1997

[Nonhistone chromosome proteins HMG1 and HMG2 stabilize one of the sequence-specific complexes, formed on the promotor of human retroposons of the ALU-family of other nuclear proteins].

Human retroposons of the Alu family have an internal promoter for RNA polymerase III also present in tRNA genes, but Alu are poorly transcribed by this polymerase in somatic cells in vivo, which is probably due to an efficient system of repression of the Alu transcription. The key control promoter element of the tRNA genes, known as B-box, binds basal transcription factor TFIIIC2, which initiates assembly of the full transcription complex. Previously we identified several human nuclear factors which bind to Alu Subregion covering B-box and the adjacent sequences, which can be involved in repression of Alu transcription. In this study we identified one factor, F1, as HMF1/2 and another factor, F2, as TFIIIC2-like protein reacting with B-box. Both the factors are not alu-specific, whereas the third identified Alu-binding factor, F3, seems to be specific for the Alu-specific subsequence located just downstream from B-box and can discriminate evolutionary "young" and "old" Alu subfamilies producing different numbers of transcripts in vivo. Although HMG1/2 binds to Alu in a sequence-unspecific manner, the proteins are capable of stabilizing the sequence-specific complex F3 which can be functionally significant. We believe that the factors binding to Alu B-box subregion interfere somehow with the functions of the basal factor TFIIIC2 and, hence, can be co-repressors of Alu transcription by RNA polymerase III.

DNA-Binding Proteins↗
PubMed1999

N-terminal and C-terminal modifications affect folding, release from the ribosomes and stability of in vitro synthesized proteins.

Important aspects of translation are release and folding of the synthesized protein into its three-dimensional structure. Studies from our group indicated that during in vitro protein synthesis a large portion of full-length polypeptides apparently accumulated as peptidyl-tRNA on ribosomes. We have also shown that some proteins though released in biologically active form may be inactivated without being degraded. These experiments were carried out by coupled transcription/translation using an Escherichia coli extract in which eukaryotic or prokaryotic test proteins were synthesized from their coding sequence inserted into specific plasmids. Experiments described here were designed to analyze the effects of N-terminal and C-terminal modifications of the coding sequence on the ribosomal release/termination process and on the stability of the newly synthesized protein. Elimination of the leader sequence in two proteins tested, mitichondrial rhodanese and bacterial beta-lactamase, caused an increase in the percentage of polypeptides released from the ribosomes relative to total synthesis. Conversely, an N-terminal extension such as a histidine-lag impaired the ribosomal release process. Also, a hydrophobic N-terminal modification of the synthesized protein reduced release of newly formed protein from the ribosomes. A C-terminal extension of the coding sequence for rhodanese by one amino acid decreased the percentage released polypeptide and furthermore affected the stability of the in vitro formed protein. We propose that a regulatory mechanism exists by which N-terminal and C-terminal sequences of a newly synthesized protein have feed-back effects on the termination factor-mediated release and on the stability of the native three-dimensional structure.

Cell-Free System↗
PubMed1985

Mitotic stability of yeast chromosomes: a colony color assay that measures nondisjunction and chromosome loss.

A colony color assay that measures chromosome stability is described and is used to study several parameters affecting the mitotic maintenance of yeast chromosomes, including ARS function, CEN function, and chromosome size. A cloned ochre-suppressing form of a tRNA gene, SUP11, serves as a marker on natural and in vitro-constructed chromosomes. In diploid strains homozygous for an ochre mutation in ade2, cells carrying no copies of the SUP11 gene are red, those carrying one copy are pink, and those carrying two or more copies are white. Thus, the degree of red sectoring in colonies reflects the frequency of mitotic chromosome loss. The assay also distinguishes between chromosome loss (1:0 segregation) and nondisjunction (2:0 segregation). The most dramatic effect on improving mitotic stability is caused by increasing chromosome size. Circular chromosomes increase in stability through a size range up to approximately 100 kb, but do not continue to be stabilized above this value. However, linear chromosomes continue to increase in mitotic stability throughout the size range tested (up to 137 kb). It is possible that the mitotic stability of linear chromosomes is proportional to chromosome length, up to a plateau value that has not yet been reached in our synthetic constructions.

Chromosomes↗
PubMed1996

Regulation of gene expression in chloroplasts of higher plants.

Chloroplasts contain their own genetic system which has a number of prokaryotic as well as some eukaryotic features. Most chloroplast genes of higher plants are organized in clusters and are cotranscribed as polycistronic pre-RNAs which are generally processes into many shorter overlapping RNA species, each of which accumulates of steady-state RNA levels. This indicates that posttranscriptional RNA processing of primary transcripts is an important step in the control of chloroplast gene expression. Chloroplast RNA processing steps include RNA cleavage/trimming, RNA splicing, ENA editing and RNA stabilization. Several chloroplast genes are interrupted by introns and therefore require processing for gene function. In tobacco chloroplasts, 18 genes contain introns, six for tRNA genes and 12 for protein-encoding genes. A number of specific proteins and RNA factors are believed to be involved in splicing and maturation of pre-RNAs in chloroplasts. Processing enzymes and RNA-binding proteins which could be involved in posttranscriptional steps have been identified in the last several years. Our current knowledge of the regulation of gene expression in chloroplasts of higher plants is overviewed and further studies on this matter are also considered.

Chloroplasts↗
PubMed2006

How ribosomes make peptide bonds.

Ribosomes are molecular machines that synthesize proteins in the cell. Recent biochemical analyses and high-resolution crystal structures of the bacterial ribosome have shown that the active site for the formation of peptide bonds--the peptidyl-transferase center--is composed solely of rRNA. Thus, the ribosome is the largest known RNA catalyst and the only natural ribozyme that has a synthetic activity. The ribosome employs entropic catalysis to accelerate peptide-bond formation by positioning substrates, reorganizing water in the active site and providing an electrostatic network that stabilizes reaction intermediates. Proton transfer during the reaction seems to be promoted by a concerted shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate.

Binding Sites↗
PubMed2004

The tRNA-like structure of Turnip yellow mosaic virus RNA is a 3'-translational enhancer.

Many positive stand RNA viral genomes lack the poly(A) tail that is characteristic of cellular mRNAs and that promotes translation in cis. The 3' untranslated regions (UTRs) of such genomes are expected to provide similar translation-enhancing properties as a poly(A) tail, yet the great variety of 3' sequences suggests that this is accomplished in a range of ways. We have identified a translational enhancer present in the 3' UTR of Turnip yellow mosaic virus (TYMV) RNA using luciferase reporter RNAs with generic 5' sequences transfected into plant cells. The 3' terminal 109 nucleotides comprising the tRNA-like structure (TLS) and an upstream pseudoknot (UPSK) act in synergy with a 5'-cap to enhance translation, with a minor contribution in stabilizing the RNA. Maximum enhancement requires that the RNA be capable of aminoacylation, but either the native valine or engineered methionine is acceptable. Mutations that decrease the affinity for translation elongation factor eEF1A (but also diminish aminoacylation efficiency) strongly decrease translational enhancement, suggesting that eEF1A is mechanistically involved. The UPSK seems to act as an important, though nonspecific, spacer element ensuring proper presentation of a functional TLS. Our studies have uncovered a novel type of translational enhancer and a new role for a plant viral TLS.

3' Untranslated Regions↗