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At least 703 records · Page 39Linked to original sources
PubMed1996

Variant minihelix RNAs reveal sequence-specific recognition of the helical tRNA(Ser) acceptor stem by E.coli seryl-tRNA synthetase.

Aminoacylation rate determinations for a series of variant RNA minihelix substrates revealed that Escherichia coli seryl-tRNA synthetase (SerRS) recognizes the 1--72 through 5--68 base pairs of the E.coli tRNA(Ser) acceptor stem with the major recognition elements clustered between positions 2--71 and 4--69. The rank order of effects of canonical base pair substitutions at each position on kcat/Km was used to assess the involvement of major groove functional groups in recognition. Conclusions based on the biochemical data are largely consistent with the interactions revealed by the refined structure of the homologous Thermus thermophilus tRNA(Ser)-SerRS complex that Cusack and colleagues report in the accompanying paper. Disruption of an end-on hydrophobic interaction between the major groove C5(H) of pyrimidine 69 and an aromatic side chain of SerRS is shown to significantly decrease kcat/Km of a minihelix substrate. This type of interaction provides a means by which proteins can recognize the binary information of 'degenerate' sequences, such as the purine-pyrimidine base pairs of tRNA(Ser). The 3--70 base pair is shown to contribute to recognition by SerRS even though it is not contacted specifically by the protein. The latter effect derives from the organization of the specific contacts that SerRS makes with the neighboring 2--71 and 4--69 acceptor stem base pairs.

Base Sequence↗
PubMed1976

Transfer RNA and aminoacyl tRNA synthetases in hormone dependent and independent mammary tumors of GR mice: I. Comparative study of the amino acid accepting capacity of the tRNA's in the presence of the homologous and heterologous enzymes.

Transfer RNA and aminoacyl tRNA synthetases were obtained from hormone dependent and independent mammary tumors of GR mice. We have studied the possible changes in the tRNA level by comparing the specific activities of the hormone dependent and independent mammary tumor tRNA's, but no differences were observed. Differences were found in the ability of enzymes from normal male GR mice liver, independent mammary tumor and dependent mammary tumor to cross-react with heterologous tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Threonyl-tRNA synthetase from yeast: aminoacylation of tRNA on its non-accepting 3'-terminal hydroxyl group and its behaviour in enzyme-catalyzed deacylation.

Methods have been developed by which tRNA Thr may be aminoacylated at the normally non-accepting 3'-terminal ribose OH. Two of the methods utilize the mischarging ability of the synthetases under special conditions of low salt concentration and presence of organic solvents. The third method demonstrates for the first time that for some synthetases the 2',3' specificity may be manipulated by use of similar special conditions. In the case of threonyl-tRNA synthetase, Thr-tRNAThr-C-C-A(3'd) has been synthesised by this method. The behaviour of threonyl esters of tRNAThr-C-C-A, tRNAThr-C-C-A(2'd) and tRNA Thr-C-C-A-(3'd) in the free enzyme-catalyzed deacylation has been studied and the results indicate that the cis diol functional group is necessary for this hydrolysis. The position on the terminal ribose from which the amino acid is removed in this reaction remains to be identified.

Adenosine Monophosphate↗
PubMed1981

Partial digestion of tRNA--aminoacyl-tRNA synthetase complexes with cobra venom ribonuclease.

Transfer RNA molecules have been labeled with 32P at the 5' or 3' end and digested with cobra venom ribonuclease, which preferentially cuts double-stranded regions. The products of yeast tRNAPhe and tRNAVal were analyzed by high-resolution gel electrohporesis. In the free state, these tRNAs were cut predominantly in the acceptor and anticodon stems. Minor cuts occurred in the T psi stem in tRNAVal. The topography of zones interacting with their cognate synthetases was studied by determining the tRNA regions shielded by protein. Nearly 100% protection was found in the anticodon and acceptor stem of tRNAVal, while in tRNAPhe only the stem of the anticodon was protected. Noncognate interactions between tRNAPhe and tryptophanyl-tRNA synthetase from beef pancreas were examined. The beef enzyme did not protect tRNAPhe despite the fact that efficient misaminoacylation occurred. The pattern of shielding obtained for each tRNA-synthetase complex was compared with the results of direct ultraviolet cross-linking experiments with these complexes.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Gram-scale purification of methionyl-tRNA and tyrosyl-tRNA synthetases from Escherichia coli.

A procedure is presented for the purification of 3 g of homogeneous methionyl-tRNA synthetase and 1 g of homogeneous tyrosyl-tRNA synthetase from 50 kg batches of Escherichia coli. The procedure permits the isolation of many enzymes simultaneously and the elution positions of seven other aminoacyl-tRNA synthetases, catalase, rhodanese, phosphofructokinase, elongation factor Tu and cytochrome b-562 are indicated. The problems of extraction work on this scale are discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1999

Complementarity between 3' terminal nucleotides of tRNA and primer binding site is a major determinant for selection of the tRNA primer used for initiation of HIV-1 reverse transcription.

The initiation of reverse transcription of human immunodeficiency virus type 1 (HIV-1) exclusively utilizes tRNALys,3 as a primer. Previous studies have shown that HIV-1 could use alternative tRNAs, such as tRNAIle or tRNAHis, to initiate reverse transcription only if the primer binding site (PBS) was made complementary to the 3' terminal 18 nucleotides of the cognate tRNA. However, upon in vitro culture, the viruses with a PBS complementary to the alternative tRNAs rapidly reverted to generate a PBS complementary to tRNALys,3. To investigate the process of reversion, we have constructed defective proviral genomes that contain a PBS complementary to tRNAIle or tRNAHis. The genomes contain the gene for xanthine-guanosine phosphoribosyl transferase (gpt) in place of env. Cotransfection of these proviral genomes with a plasmid-encoding vesicular stomatitis virus G protein (VSV-G) results in viruses that undergo a single round of HIV-1 infection; successful infections are scored as cells resistant to the drug mycophenolic acid. Using this single-round infection system, we demonstrated that HIV-1 with a PBS complementary to tRNAIle or tRNAHis is three- to fivefold less efficient in replication as measured by production of drug-resistant cell colonies compared to the wild-type virus. These viruses predominantly used the cognate tRNA as primer in their initial round of replication, although we did obtain a single cell colony in which the PBS was complementary to tRNALys,3. Using an HIV-1 provirus with a PBS complementary to yeast tRNAPhe, we established a single-round infection system in which the infectivity of this mutant HIV-1 relies on transfected yeast tRNAPhe. The results of our studies suggest that the mechanism for selection of the tRNA primer for initiation of reverse transcription relies primarily on the complementarity between the tRNA primerthe PBS.

Binding Sites↗
PubMed1984

Covalent crosslinking of Escherichia coli phenylalanyl-tRNA and valyl-tRNA to the ribosomal A site via photoaffinity probes attached to the 4-thiouridine residue.

tRNAPhe and tRNAVal of Escherichia coli were derivatized at the S4U8 position with p-azidophenacyl and p-azidophenacylacetate photoaffinity probes. The modified tRNAs could still function efficiently in all of the partial reactions of protein synthesis except for an approximately sevenfold decrease in the rate of translocation. Irradiation (310 to 340 nm) of probe-modified Phe-tRNA or Val-tRNA placed in the ribosomal A site led to crosslinking that was totally dependent on irradiation, the presence of the azido group on the probe, mRNA, and elongation factor Tu (EFTu). Prephotolysis of the modified tRNA abolished crosslinking, but prephotolysis of the ribosomes and factors had little effect. Crosslinking was efficiently quenched by mercaptoethanol or dithiothreitol, demonstrating accessibility of the probe to solvent. Use of GDPCP in place of GTP also blocked crosslinking, probably because of the retention of EFTu on the ribosome. Crosslinking with the p-azidophenacyl acetate (12 A) probe was only half as efficient as with the p-azidophenacyl (9 A) probe, and this ratio was not changed by varying Mg2+ from 5 to 15 mM. The crosslink was from a functional A site, since AcPhePhe-tRNA at the A site could be crosslinked, and it was A site-specific, because neither translocation nor direct non-enzymatic P site binding yielded any significant covalent product. The crosslink was to ribosomal protein(s) of the 30 S subunit. No other ribosomal component was crosslinked. Identification of the protein crosslinked is described in the accompanying paper.

Affinity Labels↗
PubMed1991

Informational redundancy of tRNA(4Ser) and tRNA(7Ser) genes in Drosophila melanogaster and evidence for intergenic recombination.

Variant tRNA genes have been widely observed in multicellular eukaryotes. Recent biochemical studies have shown that some of them are expressed in a tissue- or a stage-specific manner. These findings would thus imply that certain modified tRNAs may be crucial for the development of the organism. Using Drosophila melanogaster as a model, we have taken a combined genetic and molecular approach to examine critically the possible biological functions of tRNA(4, 7Ser) genes. We showed that at least 50% of the total templates can be deleted from the genome without inducing abnormal phenotypes such as Minute, or a decrease in viability. In addition, two of the tRNASer variant genes that are unique in sequence are also completely dispensable. This strongly implies that even though they may be expressed in vivo, they play no essential role in the development of the fruitfly. By comparison with some of the corresponding tRNA genes in another sibling species, Drosophila erecta, our results suggest strongly that the variants are products non-reciprocal exchanges among the tRNA(4, 7Ser), genes. Such intergenic recombination events may have a major influence in the concerted evolution of the two gene families.

Animals↗
PubMed1979

Transcription of cloned tRNA genes and the nuclear partitioning of a tRNA precursor.

The transcription of transfer RNA genes (tDNAs) and processing of the transcripts have been studied by injecting cloned tDNAs into Xenopus oocyte nuclei. Three main conclusions can be drawn. First, eucaryotic nuclear tRNA genes, but neither procaryotic nor mitochondrial tRNA genes, are expressed in injected oocytes. While both nematode and yeast tDNAS direct the synthesis of authentic tRNAs, neither E. coli tDNA nor human mitochondrial tDNAs support the synthesis of defined tRNAs when injected into oocytes. Second, competition experiments with co-injected 5S genes and inhibition experiments with alpha-amanitin show that injected tDNAs are transcribed by RNA polymerase III. Third, oocytes injected with a nematode tDNA synthesize a tRNA precursor which is processed post-transcriptionally by removal of a 5' leader sequence. This precursor is found exclusively in the nucleus and is processed in the nucleus before the mature tRNA enters the cytoplasm.

Animals↗
PubMed1981

Mutants of Su+7 tRNA include a functional tRNA with an altered T pseudo uracil CG sequence.

Su+7 tRNA, the amber-suppressing anticodon mutant of tRNA(Trp) (E. coli), also relaxes stringent control. Further mutant Su+7 tRNAs have been isolated that have lost this regulatory effect. The sequence changes we have observed are a unique set. They include partially active suppressor tRNAs with a C to A change in the T pseudo uracil CG common sequence and in the anticodon stem. The activity of the T pseudo uracil CG and anticodon-stem mutants suggests that neither sequence is essential for ribosomal function. The third mutated site, a D-stem alteration, dramatically disturbs tRNA maturation. The mutations add to our knowledge of tRNA function, particularly with regard to the dispensability of the T pseudo uracil CG sequence for activity at the ribosomal A site.

Base Sequence↗
PubMed1989

Localization of three DNA segments encompassing tRNA genes to human chromosomes 1, 5, and 16: proposed mechanism and significance of tRNA gene dispersion.

The chromosomal locations of three cloned human DNA fragments encompassing tRNA genes have been determined by Southern analysis of human-rodent somatic cell hybrid DNAs with subfragments from these cloned genes and flanking sequences used as hybridization probes. These three DNA segments have been assigned to human chromosomes 1, 5, and 16, and homologous sequences are probably located on chromosome 14 and a separate locus on chromosome 1. These studies, combined with previous results, indicate that tRNA genes and pseudogenes are dispersed on at least seven different human chromosomes and suggest that these sequences will probably be found on most, if not all, human chromosomes. Short (8-12 nucleotide) direct terminal repeats flank many of the dispersed tRNA genes. The presence of these flanking repeats, combined with the dispersion of tRNA genes throughout the human genome, suggests that many of these genes may have arisen by an RNA-mediated retroposition mechanism. The possible functional significance of this gene dispersion is considered.

Animals↗
PubMed2005

Porphyrins and porphines bind strongly and specifically to tRNA, precursor tRNA and to M1 RNA and inhibit the ribonuclease P ribozyme reaction.

Porphyrins and porphines strongly inhibit the action of the RNA subunit of the Escherichia coli ribonuclease P (M1 RNA). Meso-tetrakis(N-methyl-pyridyl)porphine followed linear competitive kinetics with pre-tRNA(Gly1) from E. coli as variable substrate (Ki 0.960 microM). Protoporphyrin IX showed linear competitive inhibition versus pre-tRNA(Gly1) from E. coli (Ki 1.90 microM). Inhibition by meso-tetrakis[4-(trimethylammonio)phenyl]porphine versus pre-tRNA(Gly1) from E. coli followed non-competitive kinetics (Ki 4.1 microM). The porphyrins bound directly to E. coli tRNAVal, E. coli pre-tRNAGly1 and M1 RNA and dissociation constants for the 1:1 complexes were determined using fluorescence spectroscopy. Dissociation constants (microM) against E. coli tRNAVal and E. coli pre-tRNAGly were: meso-tetrakis(N-methyl-pyridyl)porphine 1.21 and 0.170; meso-tetrakis[4-(trimethylammonio)phenyl]porphine, 0.107 and 0.293; protoporphyrin IX, 0.138 and 0.0819. For M1 RNA, dissociation constants were 32.8 nM for meso-tetrakis(N-methyl-pyridyl)porphine and 59.8 nM for meso-tetrakis[4-(trimethylammonio)phenyl]porphine and excitation and emission spectra indicate a binding mode with strong pi-stacking of the porphine nucleus and base pairs in a rigid low-polarity environment. Part of the inhibition of ribonuclease P is from interaction with the pre-tRNA substrate, resulting from porphyrin binding to the D-loop/T-loop region which interfaces with M1 RNA during catalysis, and part from the porphyrin binding to the M1 RNA component.

Escherichia coli↗
PubMed2004

Modified nucleotides in tRNA(Lys) and tRNA(Val) are important for translocation.

Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.

Protein Biosynthesis↗
PubMed2002

The minimal tRNA: unique structure of Ascaris suum mitochondrial tRNA(Ser)(UCU) having a short T arm and lacking the entire D arm.

The tertiary structure of Ascaris suum mitochondrial tRNA(Ser)(UCU) was examined by nuclear magnetic resonance analysis using its transcript, since tRNA(Ser)(UCU), lacking the D arm and possessing a truncated T arm, is the shortest of all the known tRNAs. Most basepairs in the proposed secondary structure of tRNA(Ser)(UCU) were shown to exist, but the connector region comprising the truncated D loop and the extra loop was flexible. This flexibility, would enable adjustment of the mutual distance between the 3'-terminus and the anticodon consistent with that of usual tRNAs. Thus, tRNA(Ser)UCU appears to function in a similar way to that of usual tRNAs in the ribosome.

Animals↗
PubMed1976

Toward an understanding of the formylation of initiator tRNA methionine in prokaryotic protein synthesis. I. In vitro studies of the 30S and 70S ribosomal-tRNA complex.

Formation of the 30S-tRNA initiation complex of Escherichia coli with nonformylated initiator tRNA is stimulated by all three initiation factors and is messenger dependent, whereas the complex formation involving the 70S ribosomes is strongly inhibited by initiation factors when the nonformylated species is used. When the 30S-Met-tRNAfMet complex is first formed and the 50S ribosomal subunit added subsequently, there is no significant inhibition by initiation factors and the nonformylated initiator tRNA is puromycin reactive. This leads to the conclusion that the formylation of the methionyl initator tRNA is only obligatory when polypeptide synthesis is initiated by nondissociated 70S ribosomes.

Binding Sites↗
PubMed2000

Escherichia coli methionyl-tRNA formyltransferase: role of amino acids conserved in the linker region and in the C-terminal domain on the specific recognition of the initiator tRNA.

The formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in eubacteria. We are studying the molecular mechanisms of recognition of the initiator tRNA by Escherichia coli MTF. MTF from eubacteria contains an approximately 100-amino acid C-terminal extension that is not found in the E. coli glycinamide ribonucleotide formyltransferase, which, like MTF, use N(10)-formyltetrahydrofolate as a formyl group donor. This C-terminal extension, which forms a distinct structural domain, is attached to the N-terminal domain through a linker region. Here, we describe the effect of (i) substitution mutations on some nineteen basic, aromatic and other conserved amino acids in the linker region and in the C-terminal domain of MTF and (ii) deletion mutations from the C-terminus on enzyme activity. We show that the positive charge on two of the lysine residues in the linker region leading to the C-terminal domain are important for enzyme activity. Mutation of some of the basic amino acids in the C-terminal domain to alanine has mostly small effects on the kinetic parameters, whereas mutation to glutamic acid has large effects. However, the deletion of 18, 20, or 80 amino acids from the C-terminus has very large effects on enzyme activity. Overall, our results support the notion that the basic amino acid residues in the C-terminal domain provide a positively charged channel that is used for the nonspecific binding of tRNA, whereas some of the amino acids in the linker region play an important role in activity of MTF.

Amino Acid Sequence↗
PubMed1979

A factor in serum and amniotic fluid is a substrate for the tRNA-modifying enzyme tRNA-guanine transferase.

Q factor, a substance found in animal serum that enables cultured mammalian cells (L-M) to produce tRNA containing queuine (the base of "nucleoside Q", queuosine), has been purified to homogeneity from bovine amniotic fluid. Q factor causes the appearance of Q-containing tRNAAsp in the L-M cells cultivated in serum-free medium, and this was used as an assay to monitor the purification of Q factor. Q factor is a competitive inhibitor of guanine for rabbit reticulocyte tRNA-guanine trnsferase, with a K1 of 4.5 x 10(-8) M. Q factor is inactivated in both the L-M cell and tRNA-guanine transferase assays by treatment with periodate or cyanogen bromide, both of which react with queuine. In L-M cells, nearly complete conversion of Q-free to Q-containing tRNAAsp is observed within 24 hr after addition of pure Q factor to the medium; actinomycin D, cycloheximide, and cycloleucine, inhibitors of RNA synthesis, protein synthesis, and nucleic acid methylation, respectively, do not inhibit this conversion. The product of the reaction, catalyzed by pure rabbit reticulocyte tRNA-guanine transferase, between Q factor and rabbit reticulocyte Q-free tRNAHis is chromatographyically indistinguishable from Q-containing tRNAHis.

Amniotic Fluid↗