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 739 records · Page 41Linked to original sources
PubMed1991

Pathway and stability of protein folding.

We describe an experimental approach to the problem of protein folding and stability which measures interaction energies and maps structures of intermediates and transition states during the folding pathway. The strategy is based on two steps. First, protein engineering is used to remove interactions that stabilize defined positions in barnase, the RNAse from Bacillus amyloliquefaciens. The consequent changes in stability are measured from the changes in free energy of unfolding of the protein. Second, each mutation is used as a probe of the structure around the wild-type side chain during the folding process. Kinetic measurements are made on the folding and unfolding of wild-type and mutant proteins. The kinetic and thermodynamic data are combined and analysed to show the role of individual side chains in the stabilization of the folded, transition and intermediate states of the protein. The protein engineering experiments are corroborated by nuclear magnetic resonance studies of hydrogen exchange during the folding process. Folding is a multiphasic process in which alpha-helices and beta-sheet are formed relatively early. Formation of the hydrophobic core by docking helix and sheet is (partly) rate determining. The final steps involve the forming of loops and the capping of the N-termini of helices.

Amino Acid Sequence↗
PubMed2003

[Establishment of a cell line with antisense-blocked POLH and the role of POLH in alkylating agent MNNG induced nontargeted mutagenesis].

OBJECTIVE: To investigate the function of POLH(polymerase eta) through establishment of the POLH gene-blocked cell line FL-POLH(-). METHODS: A mammalian expression vector expressing antisense POLH gene fragment pMAMneo-amp-POLHA (-) was constructed by cloning the 1473 - 2131 fragment of POLH gene into the mammalian expression vector pMAMneo-amp(-) in antisense orientation. The FL cells were transfected with this antisense RNA expressing vector and selected by G418. The mutation assay was conducted using the shuttle plasmid pZ189. RESULT: The spontaneous mutation frequency of SupF tRNA gene in the plasmid replicated in the FL-POLH(-) was 13.5 x 10(-4), while it was 4.9x10(-4) and 3.7x10(-4) in the control cells FL and FL-M, respectively. The nontargeted mutation frequency of SupF tRNA gene decreased in the plasmid replicated in these cell lines pretreated with MNNG. CONCLUSION: POLH plays an important role in maintenance of genetic stability and genesis of nontargeted mutation.

Antisense Elements (Genetics)↗
PubMed2006

Transcriptional activation of a moderately expressed tRNA gene by a positioned nucleosome.

All of the members of a tRNA1(Gly) multigene family from the mulberry silkworm, Bombyx mori, have identical coding regions and consequently identical internal promoter elements, but are transcribed at different levels. A moderately expressed copy, tRNA1(Gly)-4 from within this multigene family, which was transcribed to 30-50% of the highly transcribed gene copies harboured two typical TATAA box sequences in the 5' upstream region at positions -27 nt and -154 nt with respect to the +1 nt of mature tRNA. Deletion of the distal TATAA sequence at -154 nt brought down the transcription more than 70%, whereas mutation of the proximal element did not affect transcription. tRNA1(Gly)-4 could be readily assembled into chromatin, with a positioned nucleosome in the upstream region, and the assembled nucleosome formed stable complexes with the transcription factors TFIIIC and TFIIIB. Organization of the gene into nucleosomes also enhanced transcription significantly above that of the naked DNA, reaching transcription levels comparable with those of the highly transcribed copies. This nucleosome-mediated enhancement in transcription was absent when the distal TATAA sequences were deleted, whereas mutation of the proximal TATAA element showed no effect. In the absence of the distal TATAA sequences, assembly into the nucleosome inhibited transcription of tRNA1(Gly)-4. TFIIIB bound directly through the distal TATAA sequence at -154 nt and the positioned nucleosome facilitated its interaction with TFIIIC. The direct binding of TFIIIB to the DNA provided anchoring of the factor to the template DNA which conferred a higher stability on the TFIIIB-TFIIIC-DNA complex. We have proposed a novel mechanism for the nucleosome-mediated stimulation of pol III (RNA polymerase III) transcription of tRNA genes, a model not presented previously.

Animals↗
PubMed1994

Substrate specificity of tRNA (adenine-1-)-methyltransferase from Thermus thermophilus HB27.

tRNA (adenine-1-)-methyltransferase was purified to homogeneity from an extreme thermophile, Thermus thermophilus HB27, by several steps of column chromatographies. The molecular weight of this enzyme was about 60,000 as analyzed by SDS polyacrylamide gel electrophoresis. Km for E. coli tRNA(2Glu) was 100 nM and that for the methyl group donor, S-adenosyl-L-methionine, was 7.8 microM. The substrate specificity of the enzyme was investigated by using T7 RNA polymerase transcripts and tRNA fragments obtained by partial digestion with RNases. The enzyme was able to transfer the methyl group to the 3'-half fragment of E. coli initiator tRNA, however, the extent of methylation was elevated by more than five times when the 5'-half fragment was added and annealed to the 3'-half. This indicates that the main recognition site of the enzyme is within the 3'-half region of tRNA molecule, while the tertiary interaction between the T-loop and the D-loop is very effective for the adequate methylation reaction.

Base Sequence↗
PubMed1993

Aminoacylation of RNA oligonucleotides: minimalist structures and origin of specificity.

Aminoacyl tRNA synthetases are divided into two unrelated classes of ten enzymes each. Members from each class specifically aminoacylate small RNA oligonucleotides lacking anticodon sequences. Duplex structures with only four base pairs stabilized by RNA tetraloop motifs are active. Atomic groups on bases and ribose 2'-hydroxyl groups in the RNA minor groove make functional contacts that are essential for aminoacylation and provide the high specificity. A system for specific aminoacylation of small RNA oligonucleotides that is based on sequences proximal to the amino acid attachment site may reflect the small sizes of early synthetases.

Acetylation↗
PubMed1974

Thermal death of temperature-sensitive lysyl- and tryptophanyl-transfer ribonucleic acid synthetase mutants of Bacillus subtilis: effect of culture medium and developmental stage.

The growth of thermosensitive Bacillus subtilis lysyl- and tryptophanyl-transfer ribonucleic acid synthetase mutants (lysS1 and trypS1) (l-lysine:transfer ribonucleic acid [tRNA] ligase [AMP], EC 6.1.1.6; and l-tryptophan:tRNA ligase [AMP], EC 6.1.1.2) was terminated when exponential phase cells were shifted from 30 to 43 C in a rich medium. Under these conditions, the temperature-inhibited cells undergo thermal death; they rapidly lose their ability to form colonies at 30 C. Another lysyl-tRNA synthetase mutant (lysS2) is refractory to thermal death even though its growth is inhibited at 43 C. The thermal death response of the lysS1 mutant is affected by the stage of cell development. At periods in spore outgrowth and sporogenesis these cells become refractory to thermal death. The refractory state does not result from the production of an inhibitor, or from the degradation of an activator of thermal death. However, culture medium composition does modify the thermal death response. Rich media enhance the effect, and no thermal death occurs in the lysS1 strain grown in a minimal medium. Temperature-sensitive cells can grow in a lysine- (0.25 mM) or tryptophan- (0.25 mM) supplemented minimal medium at 43 C, but amino acid concentrations of 25 mM only transiently protect trypS1 and lysS1 cells from thermal death in a rich medium. Osmotic agents such as sucrose (0.5 M) and NaCl (0.34 M) completely prevent thermal death in the lysS1 strain, although growth is still arrested. On solid media, sucrose stabilized lysS1 cells can form colonies at the restrictive temperature, but neither sucrose (0.5 M) nor NaCl (0.34 M) stabilized the lysS1 enzyme in vitro. Chloramiphenicol increased the rate of thermal death of the lysS1 strain but decreased the thermal death response of the trypS1 mutant. Considering the nature of the enzyme defect in the lysS1 strain, the common genetic origin of the spore and vegetative lysyl-tRNA synthetase, and the protective effects exerted by lysine and osmotic agents, it is tentatively concluded that thermal death results from irreversible inactivation of the mutant gene product. According to this hypothesis, either the lysS1 enzyme is altered during sporogenesis or some physiological or structural aspect of this developmental phase can stabilize the mutant phenotype and thereby rescue cells from thermal death.

Alkaline Phosphatase↗
PubMed1999

Synthesis of aspartyl-tRNA(Asp) in Escherichia coli--a snapshot of the second step.

The 2.4 A crystal structure of the Escherichia coli aspartyl-tRNA synthetase (AspRS)-tRNA(Asp)-aspartyl-adenylate complex shows the two substrates poised for the transfer of the aspartic acid moiety from the adenylate to the 3'-hydroxyl of the terminal adenosine of the tRNA. A general molecular mechanism is proposed for the second step of the aspartylation reaction that accounts for the observed conformational changes, notably in the active site pocket. The stabilization of the transition state is mediated essentially by two amino acids: the class II invariant arginine of motif 2 and the eubacterial-specific Gln231, which in eukaryotes and archaea is replaced by a structurally non-homologous serine. Two archetypal RNA-protein modes of interactions are observed: the anticodon stem-loop, including the wobble base Q, binds to the N-terminal beta-barrel domain through direct protein-RNA interactions, while the binding of the acceptor stem involves both direct and water-mediated hydrogen bonds in an original recognition scheme.

Anticodon↗
PubMed2003

The pathogenic U3271C human mitochondrial tRNA(Leu(UUR)) mutation disrupts a fragile anticodon stem.

The U3271C mutation affecting the human mitochondrial transfer RNA(Leu(UUR)) (hs mt tRNA) is correlated with diabetes and mitochondrial encephalopathies. We have explored the relationship between the structural effects of this mutation and its impact on function using chemical probing experiments and in vitro aminoacylation assays to investigate a series of tRNA constructs. Chemical probing experiments indicate that the U3271C substitution, which replaces an AU pair with a CA mispair, significantly destabilizes the anticodon stem. The introduction of a compensatory A3261G mutation reintroduces base pairing at this site and restores the structure of this domain. In fact, the anticodon stem of the A3261G/U3271C mutant appears more structured than wild-type (WT) hs mt tRNA(Leu(UUR)), indicating that the entirely AU stem of the native tRNA is intrinsically weak. The results of the chemical probing experiments are mirrored in the aminoacylation activities of the mutants. The U3271C substitution decreases aminoacylation reactivity relative to the WT tRNA due to an increase in K(m) for the pathogenic mutant. The binding defect is a direct result of the structural disruption caused by the pathogenic mutation, as the introduction of the stabilizing compensatory mutation restores aminoacylation activity. Other examples of functional defects associated with the disruption of weak domains in hs mt tRNAs have been reported, indicating that the effects of pathogenic mutations may be amplified by the fragile structures that are characteristic of this class of tRNAs.

Anticodon↗
PubMed2003

Phylogeny and self-splicing ability of the plastid tRNA-Leu group I Intron.

Group I introns are mobile RNA enzymes (ribozymes) that encode conserved primary and secondary structures required for autocatalysis. The group I intron that interrupts the tRNA-Leu gene in cyanobacteria and plastids is remarkable because it is the oldest known intervening sequence and may have been present in the common ancestor of the cyanobacteria (i.e., 2.7-3.5 billion years old). This intron entered the eukaryotic domain through primary plastid endosymbiosis. We reconstructed the phylogeny of the tRNA-Leu intron and tested the in vitro self-splicing ability of a diverse collection of these ribozymes to address the relationship between intron stability and autocatalysis. Our results suggest that the present-day intron distribution in plastids is best explained by strict vertical transmission, with no intron losses in land plants or a subset of the Stramenopiles (xanthophyceae/phaeophyceae) and frequent loss among green algae, as well as in the red algae and their secondary plastid derivatives (except the xanthophyceae/phaeophyceae lineage). Interestingly, all tested land plant introns could not self-splice in vitro and presumably have become dependent on a host factor to facilitate in vivo excision. The host dependence likely evolved once in the common ancestor of land plants. In all other plastid lineages, these ribozymes could either self-splice or complete only the first step of autocatalysis.

Eukaryota↗
PubMed1987

Probing ribosome structure using short oligodeoxyribonucleotides: the question of resolution.

The structure of ribosomal RNA in situ can be probed using short, complementary DNA oligomers. As these oligomers bind to exposed, single-stranded regions of rRNA, the stability of the hybridized complex can be assayed. Differences in binding stability between cDNA probes of similar length and composition may be indicative of the presence of competing structure, such as base-paired rRNA regions, tRNA interactions or protein interactions. In this study the degree to which such interactions can be distinguished is studied. It is found that by using suitable controls, interactions between rRNA and tRNA or rRNA can be discriminated to a resolution of one or two bases. This resolution promises to be important in delineating the higher-order structure of the rRNA.

Base Sequence↗
PubMed2003

Maintenance of the correct open reading frame by the ribosome.

During translation, a string of non-overlapping triplet codons in messenger RNA is decoded into protein. The ability of a ribosome to decode mRNA without shifting between reading frames is a strict requirement for accurate protein biosynthesis. Despite enormous progress in understanding the mechanism of transfer RNA selection, the mechanism by which the correct reading frame is maintained remains unclear. In this report, evidence is presented that supports the idea that the translational frame is controlled mainly by the stability of codon-anticodon interactions at the P site. The relative instability of such interactions may lead to dissociation of the P-site tRNA from its codon, and formation of a complex with an overlapping codon, the process known as P-site tRNA slippage. We propose that this process is central to all known cases of +1 ribosomal frameshifting, including that required for the decoding of the yeast transposable element Ty3. An earlier model for the decoding of this element proposed 'out-of-frame' binding of A-site tRNA without preceding P-site tRNA slippage.

Anticodon↗
PubMed2002

Ribosome-associated factor Y adopts a fold resembling a double-stranded RNA binding domain scaffold.

Escherichia coli protein Y (pY) binds to the small ribosomal subunit and stabilizes ribosomes against dissociation when bacteria experience environmental stress. pY inhibits translation in vitro, most probably by interfering with the binding of the aminoacyl-tRNA to the ribosomal A site. Such a translational arrest may mediate overall adaptation of cells to environmental conditions. We have determined the 3D solution structure of a 112-residue pY and have studied its backbone dynamic by NMR spectroscopy. The structure has a betaalphabetabetabetaalpha topology and represents a compact two-layered sandwich of two nearly parallel alpha helices packed against the same side of a four-stranded beta sheet. The 23 C-terminal residues of the protein are disordered. Long-range angular constraints provided by residual dipolar coupling data proved critical for precisely defining the position of helix 1. Our data establish that the C-terminal region of helix 1 and the loop linking this helix with strand beta2 show significant conformational exchange in the ms- micro s time scale, which may have relevance to the interaction of pY with ribosomal subunits. Distribution of the conserved residues on the protein surface highlights a positively charged region towards the C-terminal segments of both alpha helices, which most probably constitutes an RNA binding site. The observed betaalphabetabetabetaalpha topology of pY resembles the alphabetabetabetaalpha topology of double-stranded RNA-binding domains, despite limited sequence similarity. It appears probable that functional properties of pY are not identical to those of dsRBDs, as the postulated RNA-binding site in pY does not coincide with the RNA-binding surface of the dsRBDs.

Amino Acid Sequence↗
PubMed1978

Structural analysis of spermine and magnesium ion binding to yeast phenylalanine transfer RNA.

Refinement of the diffraction data at 2.5-A resolution from orthorhombic crystals of yeast tRNAPhe has proceeded to the point where spermine and magnesium ions can be located in the difference electron density map. Two spermine molecules are found: one is located in the major groove at one end of the anticodon stem; the other is near the variable loop and curls around phosphate 10 in a region where the polynucleotide chain takes a sharp turn. Four distinct magnesium ions have been identified: one in the anticodon loop, two in the D loop, and one coordinated with phosphates 8, 9, 11, and 12, where the polynucleotide chain is coiled. The conformation of the anticodon stem and loop is stabilized by the cations at the end of the molecule. The positions of these ions may be related to aspects of the biological activity of tRNA. The spermine and magnesium ions appear to be important in maintaining the overall folding of the tRNA molecule.

Anticodon↗
PubMed1986

Contextual constraints on codon pair usage: structural and biological implications.

Complementary DNA sequence data of 278 protein coding genes from prokaryotic systems have been analysed at the level of near neighbour codon pairs. Our analysis points out that constraints exist even at the level of near neighbour codon pairs. These constraints are in addition to those which arise due to relative levels of tRNA. Codon pairs, which in the data base have different occurrence values from their expected values, neither have common secondary structure nor do have better stabilization due to high base stacking. Our study points out that there are strong interaction between constituent codons in these codon pairs. These strongly interacting codon pairs, we suggest, are involved in the formation of three dimensional structural elements of cDNA/mRNA and interact with ribosome and thus modulate translation.

Base Sequence↗