Primary sequence of tRNA val from Escherichia coli B. I. Oligonucleotide sequences of digests of Escherichia coli tRNA val with RNase T and pancreatic RNase.
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Adenine Nucleotides↗
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Results for “RNA, Transfer, Val”
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Residual tRNA secondary structure in 'denaturing' 8M urea/TBE polyacrylamide gels: effects on electrophoretic mobility and dependency on prior chemical modification of the tRNA.
Fifteen individual species of tRNA were treated with the chemical modifiers diethylpyrocarbonate, 50% aqueous hydrazine or hydrazine/3 M NaCl. Following purification of the chemically modified material on polyacrylamide gels containing 8 M urea, variant minor bands, in addition to the expected main band, were observed for 12 of the 15 tRNAs. Characterization of the content of chemically altered bases in material recovered from such bands indicated that tRNAs containing modified nucleotides in base-paired stems were excluded from the main band and present, often in enhanced amounts, in the minor variant bands. The persistence of residual secondary structure on 8 M urea gels run at 45 degrees C and the ability of chemically modified bases to alter electrophoretic mobilities warrant caution in designing and interpreting experiments in which chemically modified RNA is isolated on gels prior to further analysis. tRNA(Val) (VAC) was unique in that modified bases in non base-paired regions, according to the cloverleaf model of secondary structure, caused exclusion from the main band. Consequently, we propose a secondary structure for partially denatured tRNA(Val) (VAC), in which these bases are located in double stranded regions of the molecule.
Base Sequence↗
The formation of pyrrolid-2-one-5-carboxylic acid at the N-terminus of immunoglobulin G heavy chain.
We propose that pyrrolid-2-one-5-carboxyl-tRNA is not involved in the initiation of protein synthesis in eukaryotic cells and that the N-terminal pyrrolid-2-one-5-carboxylic acid group of an IgG (immunoglobulin G) (that secreted by the mouse plasmacytoma Adj PC5) is formed by the enzymic cyclization of the N-terminal glutamine of the heavy chain of the completed IgG molecule and that the cyclization takes place inside the cell. We base these conclusions on the following evidence. (1) Pyrrolidonecarboxyl-tRNA was not found in incorporation experiments with rat liver preparations and [U-(14)C]-pyrrolidonecarboxylic acid, glutamic acid and glutamine, even though an incorporation extent of less than 2% of the total products could have been detected. (2) Double-labelling experiments showed that less than 8% of the nascent peptides of heavy chains (those obtained by precipitation by the antibody to Fc fragment) began with pyrrolidonecarboxylic acid. (3) Further double-labelling experiments showed that 60-66% of the heavy chains of the completed intracellular IgG molecule began with pyrrolidonecarboxylic acid after both 1 and 5h of labelling. (4) The IgG, after secretion by plasmacytoma Adj PC5, was found to have the sequence [unk]Glu- Val-Gln-Leu- at the N-termini of the heavy chains.
Amino Acid Sequence↗
[Corrective steps in amino acid activation for protein biosynthesis].
Activation of aminoacids for proteinbiosynthesis is not absolutely specific. With tRNA modified at the 3' end we were able to show, that valine misactivated by isoleucyl-tRNA synthetase is transferred to tRNA(I1e). Val-tRNA(I1e) is then hydrolyzed by the enzyme prior to release of the wrong product. For this hydrolysis the nonaccepting 3'OH of the 3'-terminal ribose is essential. The role of the 3'OH is to activate a water molecule which, in the case of valine, is inserted into the place normally occupied by the methyl group of isoleucine. Hence hydrolysis is specific for misactivated valine while the correct substrate, isoleucine, is protected by its additional methyl group.
Isoleucine-tRNA Ligase↗
Studies on fish liver protein synthesis. II. Factors influencing the aminoacylation of shark liver transfer ribonucleic acid.
The influence of buffer, pH, Mg2+, ATP, Na+, K+ and temperature on the extent and rate of aminoacyl-tRNA synthesis was studied with the shark Mustelus mento liver tRNAs and aminoacyl-tRNA synthetases. The optimum pH for the aminoacylation of tRNAMet was 8.3 and for tRNAVal 7.0. For these two tRNAs the Mg2+ optimum was related to the levels of ATP required and to the pH of the reactions. It is suggested that the Mg2+ concentration required for each aminoacylation system reflects the true conditions under which the substrate for the enzyme, the MgATP2- complex, is formed. The effect of monovalent cations was also examined. Val-tRNA synthesis was slightly stimulated up to a concentration of 50 mM NaCl (KCl). Over 100 mM salt, a rapid inhibition was observed. Met-tRNA synthesis behaves differently by being stimulated over a wide range of salt concentrations.
Acetylation↗
The aminoacyl-tRNA population of human reticulocytes.
The aminoacyl-tRNA population of human reticulocytes has been examined. These studies include: 1) determination of the levels of amino acid acceptance for 20 aminoacyl-tRNAs; 2) comparison of 20 aminoacyl-tRNAs from human reticulocytes to those of rabbit reticulocytes by reverse phase chromatography; 3) comparison of the levels of Ile- and Leu-tRNAs in fetal and adult reticulocytes; and 4) determination of the codon recognition properties of human Ala-, Asn-, His-, Leu-, Thr-, and Val-tRNAs. These studies provide evidence that the aminoacyl-tRNA population of human reticulocytes is adapted to the requirements of protein synthesis.
Adult↗
Catalytic activity of aminoacyl tRNA synthetases and its implications for the origin of life. I. Aminoacyl adenylate formation in tyrosyl tRNA synthetase.
The changes in the catalytic activity resulting from amino acid substitutions in the active site region have been theoretically modeled for tyrosyl tRNA synthetase (Tyr-RS). The catalytic activity was calculated as the differential stabilization of the transition state using electrostatic approximation. The results indicate that charged residues His45, His48, Asp78, Asp176, Asp194, Lys225, Lys230, Lys233, Arg265, and Lys268 play essential roles in catalysis of aminoacyl adenylate formation in Tyr-RS, which is in general agreement with previously known experimental data for residues 45, 48, 194, 230, and 233. These catalytic residues have also been used to search for sequence homology patterns among class I aminoacyl RSs of which HIGH and KMSKS conserved sequence motifs are well known. His45 and His48 belong to the HIGH signature sequence of class I aminoacyl tRNA synthetases (aRSs), whereas Arg265 and Lys268 can constitute a part of the KMSKS charge pattern. Lys225, Lys230, and Lys233 may be part of the conservative substitution pattern [HKR]-X(4)-[HKR]-X(2)-[HKR], and Asp194 is part of the new GSDQ motif. This demonstrates that the three dimensional charge distribution near the active site is an essential feature of the catalytic activity of aRS and that the theoretical technique used in this work can be utilized in searches for the catalytically important residues that may provide a clue for a charge residue pattern conserved in evolution. The appearance of patterns I-IV in Arg-, Gln-, Met-, Ile-, Leu-, Trp-, Val-, Glu-, Cys-, and Tyr-RS indicates that all these enzymes could have the same ancestor.
Amino Acid Sequence↗
Codon recognition patterns as deduced from sequences of the complete set of transfer RNA species in Mycoplasma capricolum. Resemblance to mitochondria.
The nucleotide sequences of the complete set of tRNA species in Mycoplasma capricolum, a derivative of Gram-positive eubacteria, have been determined. This bacterium represents the first genetic system in which the sequences of all the tRNA species have been determined at the RNA level. There are 29 tRNA species: three for Leu, two each for Arg, Ile, Lys, Met, Ser, Thr and Trp, and one each for the other 12 amino acids as judged from aminoacylation and the anticodon nucleotide sequences. The number of tRNA species is the smallest among all known genetic systems except for mitochondria. The tRNA anticodon sequences have revealed several features characteristic of M. capricolum. (1) There is only one tRNA species each for Ala, Gly, Leu, Pro, Ser and Val family boxes (4-codon boxes), and these tRNAs all have an unmodified U residue at the first position of the anticodon. (2) There are two tRNAThr species having anticodons UGU and AGU; the first positions of these anticodons are unmodified. (3) There is only one tRNA with anticodon ICG in the Arg family box (CGN); this tRNA can translate codons CGU, CGC and CGA. No tRNA capable of translating codon CGG has been detected, suggesting that CGG is an unassigned codon in this bacterium. (4) A tRNATrp with anticodon UCA is present, and reads codon UGA as Trp. On the basis of these and other observations, novel codon recognition patterns in M. capricolum are proposed. A comparatively small total, 13, of modified nucleosides is contained in all M. capricolum tRNAs. The 5' end nucleoside of the T psi C-loop (position 54) of all tRNAs is uridine, not modified to ribothymidine. The anticodon composition, and hence codon recognition patterns, of M. capricolum tRNAs resemble those of mitochondrial tRNAs.
Amino Acids↗
Analysis of isoaccepting transfer ribonucleic acid species of Bacillus subtilis: chromatographic differences between transfer ribonucleic acids from spores and cells in exponential growth.
Differences between the transfer ribonucleic acid (tRNA) of spores and exponentially growing cells of Bacillus subtilis 168 were compared by co-chromatography on reversed-phase column RPC-5. This system gave excellent resolution of isoaccepting species in 1 to 2 hr using a 200-ml gradient. Two methods were used to extract spore tRNAs, a procedure using a Braun homogenizer and a pretreatment with dithiothreitol followed by lysis with lysozyme. Where changes were observed, column elution profiles of spore tRNAs were independent of the extraction method used. Three kinds of changes between the profiles of vegetative cell tRNA and spore tRNA were observed: (i) no change; phe-, val-, ala-, asp-, ileu-, pro-, met-, fmet-, and his-tRNAs, (ii) a change in the ratio of existing peaks; gly-, tyr-, leu-, ser-, thr-, aspn-, and arg-tRNAs, and (iii) the appearance or disappearance of unique peaks; lys-, glu-, and trp-tRNAs.
Acylation↗
3-D graphics modelling of the tRNA-like 3'-end of turnip yellow mosaic virus RNA: structural and functional implications.
The tRNA-like structure of the aminoacylatable 3'-end of turnip yellow mosaic virus (TYMV) RNA was submitted to 3-D graphics modelling. A model of this structure has been inferred previously from both biochemical results and sequence comparisons which presents a new RNA folding feature, the "pseudoknot". It has been verified that this structure can be constructed without compromising accepted RNA stereochemical rules, namely base stacking and preferential 3'-endo sugar pucker. The model has aided interpretation of previous structural mapping experiments using chemical and enzymatic probes, and new accessibilities of residues could be predicted and tested. Pseudoknots have been considered as potential splice sites because they form antiparallel helical segments in a single RNA molecule. We have examined this possibility with the constructed 3-D model and could verify the hypothesis on a structural basis. The model presents a striking similarity with canonical tRNA and allows a valuable comparison between the protection patterns of yeast tRNA(Val) and tRNA-like viral RNA by cognate yeast valyl-tRNA synthetase against structural probes.
Base Sequence↗
[Amino-acylation of Escherichia coli tRNA-1-Val by phenylalanine-tRNA synthetase of yeast].
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Adenosine Triphosphate↗
Catalytic mechanism of valyl-tRNA synthetase from baker's yeast. Reaction pathway and rate-determining step in the aminoacylation of tRNAVal.
The catalytic mechanism of valyl-tRNA synthetase from baker's yeast has been investigated by pre-steady-state and steady-state kinetic measurements and end product dissociation studies. The pre-steady-state kinetics show a lag period during the early time when the reaction is started with free enzyme. The preincubation of the synthetase with tRNAVal and/or valine or preformation of Val approximately AMP leads to a progressive suppression of the lag. This lag probably reflects conformational transitions of the enzyme-substrate complex necessary for the transfer. At low pH or at a low ionic strength, the tRNAVal charging occurs much faster at the pre steady state than at the steady state. We show that after the fast transfer of valine from adenylate to tRNAVal, followed by the fast dissociation of AMP and PPi, a new adenylate is synthesized which promotes the dissociation of the nascent Val-tRNAVal. This dissociation occurs in a multistep process. First ATP and magnesium promote the ejection of the valine moiety of Val-tRNAVal from the adenylate site. A new adenylate is then synthesized which promotes, in the presence of magnesium, several state changes of the end product complex. A complex is finally generated in which the enzyme-bound Val-tRNAVal is able to exchange rapidly with a tRNAVal molecule. The free tRNAVal plays an active role in this exchange. Depending upon the experimental conditions, one of these steps can determine the steady-state rate of tRNAVal charging. The dissociations of enzyme-bound uncharged tRNAVal or aa-tRNAs substituted on the amino acid or on the tRNA parts by noncognate parts as well as the effect of the replacement of the adenylates by wrong adenylates have been investigated. It is shown that the valine and the tRNA moieties of Val-tRNAVal and the valine moiety of the adenylate are involved in this mechanism of dissociation. Finally, the rate-determining step of the reversal of tRNAVal charging at the steady-state has been investigated. It is shown that this step is the dissociation of the deacylated tRNAVal from enzyme.
Acylation↗
Maximum-entropy calculation of free energy distributions in tRNAs.
We have previously shown that the distribution function describing the probability that a biological macromolecule picked at random has a particular enthalpy value can be calculated from the temperature dependence of the heat capacity of the macromolecule. The free energy as a function of enthalpy (free energy distribution) can then be determined from the enthalpy probability distribution. In addition, the free energy distribution at an arbitrary temperature can be calculated from the free energy distribution at a reference temperature. Here we apply this approach to a family of similar macromolecules, namely a set of transfer RNAs, specifically tRNA(Phe), tRNA(Val), tRNA(Met), tRNA(Ser), tRNA(Asp) and tRNA(Ile). Using published heat-capacity data, we calculate the enthalpy probability distribution functions for all of these molecules at five different temperatures covering the range from 30 to 80 degrees C. We then use these distributions to give a reference free-energy distribution, from which the thermodynamics at any temperature can be calculated for each species. We compare the reference free-energy distribution for the five tRNAs and find that, while the overall form of the distributions is similar, the local behavior of the functions varies considerably between the species.
Entropy↗
Primary sequence of tRNA-Val-1 from Escherichia coli B. II. Isolation of large fragments by limited digestion with RNases, and overlapping of fragments to reduce the total primary sequence.
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Bacillus subtilis↗
Azospirillum lipoferum tRNAs: fractionation and identification of tRNA species and the nucleotide sequence of tRNA(Asn) (QUU).
Transfer RNAs of Azospirillum lipoferum were separated by two-dimensional gel electrophoresis and identified by aminoacylation. Thirty-six tRNA spots were resolved by this technique and twenty-six tRNA species have been identified. There are five tRNAs for Leu, four for Val, three for Pro, two each for Arg, Ile, Lys and Tyr, and one each for Ala, Asp, His, Phe, Ser and Thr. The tRNA (Asn) (QUU) was purified and its nucleotide sequence was determined. The A. lipoferum tRNA (Asn) (QUU) is 92% similar to B. subtilis tRNA (Asn) gene and two hypermodified nucleosides, queuosine (Q) and N-(9-beta-D Ribofuranosylpurine-6-YL)-carbamoyl)-threonine (t6A) are present in this tRNA.
Azospirillum↗
The case for the anticode.
The present paper will focus on the developments in our lab related to the origin of the code since the Israel meeting (1,2). Principally these items are: (a) a new set of correlations (3) which include ranked hydrophobicities of amino acids and dinucleotides; (b) binding constants (4) of Phe for the four mononucleotides; and (c) binding constants (5) of Phe, Leu, Ile, Val, and Gly for polyadenylic acid (poly A). The data continue to support a model for the origin of the code based on relationships between amino acids and their anticodons.
Amino Acids↗
Nucleotide sequence of a spinach chloroplast valine tRNA.
The nucleotide sequence of a spinach chloroplast valine tRNA (sp. chl. tRNA Val) has been determined. This tRNA shows essentially equal homology to prokaryotic valine tRNAs (58-65% homology) and to the mitochondrial valine tRNAs of lower eukaryotes (yeast and N. crassa, 61-62% homology). Sp. chl. tRNA Val shows distinctly lower homology to mouse mitochondrial valine tRNA (53% homology) and to eukaryotic cytoplasmic valine tRNAs (47-53% homology). Sp. chl. tRNA Val, like all other chloroplast tRNAs sequenced, contains a methylated GG sequence in the dihydrouridine loop and lacks unusual structural features which have been found in several mitochondrial tRNAs.
Animals↗
The larger genomic RNA of Helicoverpa armigera stunt tetravirus encodes the viral RNA polymerase and has a novel 3'-terminal tRNA-like structure.
In this paper we report the complete nucleotide sequence of the larger segment (5312 nucleotides) of the bipartite RNA genome of Helicoverpa armigera stunt tetravirus (HaSV). HaSV therefore becomes the first member of the Tetraviridae, a virus family with a host range restricted to lepidopteran insects, whose genome has been completely sequenced. HaSV RNA 1 encodes a 187K protein which includes three domains conserved in RNA-dependent RNA polymerases of RNA viruses in the alpha-like superfamily. Analysis of the replicase sequence confirms the status of the Tetraviridae as a distinct family within this superfamily, which includes animal, plant, and insect viruses, and shows the least-distantly related replicase for all three domains to be that of the hepatitis E virus. Another feature of the nonpolyadenylated HaSV genomic RNAs is a well-conserved 3'-terminal tRNA-like structure, the first such structure discerned in an animal virus. However, in contrast to the tRNA-like structures on some plant virus RNAs, the HaSV structure, which has a valine anticodon (CAU), appears to form without a pseudoknot and therefore resembles authentic tRNA(Val) more closely than do the plant viral structures. The implications of these observations for our understanding of RNA virus evolution are discussed.
3' Untranslated Regions↗