Nucleotide sequence of a region encoding subunit 6 of NADH dehydrogenase (ND6) and tRNA(Trp) in Chlamydomonas reinhardtii mitochondrial DNA.
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Amino Acid Sequence↗
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Results for “RNA, Transfer, Trp”
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Isolation and transcriptional competence of three tRNA(Trp) genes from Arabidopsis thaliana L.
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Base Composition↗
Comparison of tRNA(Trp) and tRNA(Pro) gene sequences in the chloroplast and mitochondrial genomes of oenothera.
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Base Sequence↗
An active role for tRNA in decoding beyond codon:anticodon pairing.
During transfer RNA (tRNA) selection, a cognate codon:anticodon interaction triggers a series of events that ultimately results in the acceptance of that tRNA into the ribosome for peptide-bond formation. High-fidelity discrimination between the cognate tRNA and near- and noncognate ones depends both on their differential dissociation rates from the ribosome and on specific acceleration of forward rate constants by cognate species. Here we show that a mutant tRNA(Trp) carrying a single substitution in its D-arm achieves elevated levels of miscoding by accelerating these forward rate constants independent of codon:anticodon pairing in the decoding center. These data provide evidence for a direct role for tRNA in signaling its own acceptance during decoding and support its fundamental role during the evolution of protein synthesis.
Anticodon↗
Incomplete factorial and response surface methods in experimental design: yield optimization of tRNA(Trp) from in vitro T7 RNA polymerase transcription.
We have studied the yield of Escherichia coli tRNA(Trp) obtained from in vitro T7 RNA polymerase transcription using incomplete factorial and response surface methods. Incomplete factorial experiments were first used to estimate the relative impact of six variables on the yield of tRNA(Trp). Fifteen trials were performed according to a balanced and randomized design. The correlation between observed yield and all experimental variables was identified by stepwise multiple linear regression analysis. The concentrations of T7 RNA polymerase, DNA template, NTP and MgCl2 proved to be significantly correlated with the yield of tRNA(Trp). We then optimized the yield with respect to each of these four variables simultaneously with a designed, response surface experiment based on the Hardin-Sloane minimum prediction variance algorithm. Twenty experiments were performed, in duplicate, to sample the quadratic surface relating the yield to the four significant variables. Coefficients of the quadratic function with all two-factor interactions were evaluated by stepwise regression using least squares, and significant coefficients were retained. Partial differentiation of the resulting quadratic model showed it to possess an optimum. Transcription performed at the corresponding conditions yielded 6-fold more tRNA(Trp) than the initial conditions, confirming the predictive value of the experimentally determined response surface.
Base Composition↗
Purification of hyperexpressed Bacillus subtilis tRNA(Trp) cloned in Escherichia coli.
To study the effects of evolutionary sequence changes on the molecular interactions of tRNA inside the cell, the Bacillus subtilis tRNA(Trp) gene has been cloned into Escherichia coli JM109 under the control of the lac promotor. Hyperexpression of the gene in minimal medium upon induction yielded 28% of total tRNA in the form of B. subtilis tRNA(Trp). The tRNA(Trp) gene product was purified by the use of a single Vydac C4 high-performance liquid chromatography (HPLC) matrix. This experimental system provided a valuable system for the hyperexpression and purification of a heterologous tRNA for studies in vitro. Moreover, because HPLC fractionation of the heterologous tRNA(Trp) gene product yielded multiple peaks, the system made possible an analysis of the molecular mechanisms for the transcriptional modifications of the tRNA(Trp) gene product in vivo.
Bacillus subtilis↗
Analysis of acceptor stem base pairing on tRNA(Trp) aminoacylation and function in vivo.
The role of acceptor stem base pairs in determining the identity of Escherichia coli tRNA(Trp) was examined by complementation of an E. coli strain containing a temperature-sensitive tRNA(Trp) gene (trpTts) and by monitoring aminoacylation levels in vivo. All derivatives of tRNA(Trp) containing substitutions at the first 3 base pairs in the acceptor stem complemented the trpTts mutation at the nonpermissive temperature (42 degrees C). However, three acceptor stem derivatives (tRNA(Trp)/C1.G72, tRNA(Trp)/C2.G71, and tRNA(Trp)/A3.U70) required overexpression for growth at 42 degrees C. Northern analysis of these derivatives following acid/urea gel electrophoresis showed no defects in tRNA aminoacylation at the nonpermissive temperature. Instead, these tRNAs appear to be defective in translation. This was suggested by the weak opal suppressor activities of the corresponding tRNA(UCATrp) derivatives. These results demonstrate that the three terminal acceptor stem base pairs do not contribute to the identity of tRNA(Trp). Substitution of the C1.A72 base pair in a methionine initiator tRNA containing the tryptophan anticodon and discriminator base (tRNA(CCAfMet)/G73) with A1.U72, the base pair found in tRNA(Trp), or G1.C72 resulted in the conversion of these tRNAs into tryptophan-inserting elongator tRNAs in vivo. However, changes to U1.A72 or C1.G72 in tRNA(CCAfMet)/G73 resulted in misaminoacylation and/or defects in translation. Our data indicate that the A1.U72 base pair is a context-dependent, negative identity element of tRNA(Trp).
Anticodon↗
Primer tRNA(Trp) of RSV-transformed or RAV-1-infected cells up-regulates the antiribosomal activity of gelonin.
Some ribosome-inactivating proteins (RIPs) with RNA-N-glycosidase activity on 28S rRNA require, for maximal inactivation of ribosomes, the presence of tRNA. tRNA(Trp) specifically up-regulates gelonin, the RIP from Gelonium multiflorum. The same tRNA is the primer of the reverse transcriptase of Rous sarcoma virus (RSV) and of its mutant (RAV-1) which lacks the src gene. Here we demonstrate that gelonin is more active in inhibiting endogenous protein synthesis by lysates of RSV-transformed or RAV-1-infected cells and that such increase in activity correlates with the increased amount of primer tRNA(Trp) in the cells.
Animals↗
Effects of the ligands of beef tryptophanyl-tRNA synthetase on the elementary steps of the tRNA(Trp) aminoacylation.
Tryptophanyl-tRNA synthetase catalyzed formation of Trp-tRNA(Trp) has been studied by mixing tRNA(Trp) with a preformed bis(tryptophanyl adenylate)-enzyme complex in the 0-60-ms time range, on a quenched-flow apparatus. Analyzing the data gives an association rate constant ka = (1.22 +/- 0.47) X 10(8) M-1 S-1, a dissociation rate constant kd = 143 +/- 73 S-1, and a dissociation constant Kd = 1.34 +/- 0.80 microM for tRNA(Trp). The maximum rate constant of tryptophan transfer to tRNA(Trp) is kt = 33 +/- 3 S-1. When starting the aminoacylation reaction with a mono(tryptophanyl adenylate)-enzyme complex, one obtains different kinetic profiles than when using a bis(tryptophanyl adenylate)-enzyme complex. Over a 0-400-ms time range, the monoadenylate-enzyme complex yields an apparent first-order reaction, while the bis-adenylate-enzyme complex yields a biphasic aminoacylation of tRNA(Trp). Analysis of Trp-tRNA(Trp) formation from both complexes according to simple reaction schemes shows that the dissociation of tRNA(Trp) from an enzyme subunit carrying no adenylate is 6.9-fold slower than from an enzyme subunit carrying an adenylate. The apparent rate constant of dissociation of nascent tryptophanyl-tRNA(Trp) is 4.9 S-1 in the absence of free tryptophan, which is much slower than its rate of formation (33 S-1).(ABSTRACT TRUNCATED AT 250 WORDS)
Acylation↗
tRNA(Trp) translation of leader peptide codon 12 and other factors that regulate expression of the tryptophanase operon.
Tryptophanase (tna) operon expression in Escherichia coli is induced by tryptophan. This response is mediated by features of a 319-base-pair leader region preceding the major structural genes of the operon. Translation of the coding region (tnaC) for a 24-amino-acid leader peptide is essential for induction. We have used site-directed mutagenesis to investigate the role of the single Trp codon, at position 12 in tnaC, in regulation of the operon. Codon 12 was changed to either a UAG or UGA stop codon or to a CGG arginine codon. Induction by tryptophan was eliminated by any of these changes. Studies with suppressor tRNAs indicated that tRNA(Trp) translation of codon 12 in tnaC is essential for induction of the operon. Reduction of tna expression by a miaA mutation supports a role for translation by tRNA(Trp) in regulation of the operon. Frameshift mutations and suppression that allows translation of tnaC to proceed beyond the normal stop codon result in constitutive tna operon expression. Deletion of a potential site for Rho factor utilization just beyond tnaC also results in partial constitutive expression. These studies suggest possible models for tryptophan induction of tna operon expression involving tRNA(Trp)-mediated frame shifting or readthrough at the tnaC stop codon.
Chromosome Deletion↗
Residues Lys-149 and Glu-153 switch the aminoacylation of tRNA(Trp) in Bacillus subtilis.
Tryptophanyl-tRNA synthetase (TrpRS) consists of two identical subunits that induce the cross-subunit binding mode of tRNA(Trp). It has been shown that eubacterial and eukaryotic TrpRSs cannot efficiently cross-aminoacylate the corresponding tRNA(Trp). Although the identity elements in tRNA(Trp) that confer the species-specific recognition have been identified, the corresponding elements in TrpRS have not yet been reported. In this study two residues, Lys-149 and Glu-153, were identified as being crucial for the accurate recognition of tRNA(Trp). These residues reside adjacent to the binding pocket for Trp-AMP and show phylogenic diversities in the charge on their side chains between eubacteria and eukaryotes. Single mutagenesis at Lys-149 or Glu-153 reduced the activity of TrpRS in the activation of Trp. The reduction was less than that caused by the double mutant WBHA (K149D/E153R). It is unusual that E153G had no detectable activity in the activation of Trp unless tRNA(Trp) was added to the reaction. In addition, we successfully switched the species specificity of Bacillus subtilis TrpRS recognition of tRNA(Trp). The affinity of WBHA, K149E and E153K to human tRNA(Trp) was 31-, 13.5-, and 12.9-fold greater than that of wild type B. subtilis TrpRS, respectively. Indeed WBHA and E153K were found to prefer genuine human tRNA(Trp) to their cognate eubacteria tRNA(Trp).
Acylation↗
Characterization of the tRNA(Trp) genes of Saccharomyces cerevisiae.
The purpose of this work was to examine the tRNA(Trp)-encoding genes (tRNA(Trp)) of Saccharomyces cerevisiae to gain insight as to why tRNA(Trp) amber suppressors, isolated by conventional genetic techniques, have not been reported. The results herein indicate that the haploid yeast genome contains six tRNA(Trp) genes which map to five or six chromosomes. Not only do the six genes have identical coding sequences but their introns are also identical. Gene replacement experiments indicate that five copies of tRNA(Trp) are sufficient for cell viability. Thus, mutation of one tRNA(Trp) gene to a suppressor in vivo, lowering the functional number of tRNA(Trp) genes, would not be expected to be lethal.
Base Sequence↗
Spiroplasma citri UGG and UGA tryptophan codons: sequence of the two tryptophanyl-tRNAs and organization of the corresponding genes.
From the total tRNAs of Spiroplasma citri, we isolated and purified two tRNA(Trp) species by using chromatography on an RPC-5 column followed by denaturing polyacrylamide gel electrophoresis. The sequence of the two tRNAs, as well as the sequences of the corresponding genes, were determined. One of the two tRNA(Trp) species has a CCA anticodon and is able to pair with the universal UGG tryptophan codon, while the second has a U*CA (U* is a modified uridine) anticodon and is able to pair with UGA but also with UGG in accordance with the "U:N wobble" rule. Thus, in S. citri, UGA is not a stop codon but codes for tryptophan. The two tRNA(Trp) genes, together with a third tRNA gene, tRNA(Ser) (CGA), belong to a single transcription unit. The nucleotide sequences of the two tRNA(Trp) species show 82.9% similarity. The two spiroplasmal tRNA(Trp) species can be aminoacylated by using an aminoacyl-tRNA synthetase fraction from S. citri. In contrast, the enzyme fraction from Escherichia coli aminoacylates tRNA(Trp) (CCA) but not tRNA(Trp) (U*CA).
Base Sequence↗
The chloroplast-derived trnW and trnM-e genes are not expressed in Arabidopsis mitochondria.
Depending on their genetic origin, plant mitochondrial tRNAs are classified into three categories: the "native" and "chloroplast-like" mitochondrial-encoded tRNAs and the imported nuclear-encoded tRNAs. The number and identity of tRNAs in each category change from one plant specie to another. As some plant mitochondrial trn genes were found to be not expressed, and as all Arabidopsis thaliana mitochondrial trn genes are known, we systematically tested the expression of A. thaliana mitochondrial trn genes. Both the "chloroplast-like" trnW and trnM-e genes were found to be not expressed. These exceptions are remarkable since trnW and trnM-e are expressed in the mitochondria of other land plants. Whereas we could not conclude which tRNA(Met) compensates the lack of expression of trnM-e, we showed that the cytosolic tRNA(Trp) is present in A. thaliana mitochondria, thus compensating the absence of expression of the mitochondrial-encoded trnW.
Arabidopsis↗
Alteration of transfer ribonucleic acid of Neisseria meningitidis during growth in the presence of human transferrin.
Iron-related tRNA alterations have been shown to occur in several pathogens but nothing has been reported about the effect of iron on meningococcal tRNA. The chromatographic elution profile of 3H-tryptophan-tRNA(trp) from a Neisseria meningitidis strain grown under different conditions was examined. The profile showed an early (P1) and a late (P2) eluting species of tRNA(trp), but the proportion of the two species varied under different growth conditions. The elution profile of trp-tRNA(trp) from bacteria grown in iron-sufficient Mueller Hinton broth yielded a minor P1 species and a major P2 species, whereas under iron-restricted growth induced by desferrioxamine, the pattern was one of a major P1 species and minor P2 species. Iron-restriction induced by human transferrin (HTF) resulted in almost equal amounts of the two tRNA(trp) species (P1 congruent to P2). Differences in the proportions of the tRNA species were also found between cells grown in liquid medium (P1 << P2) and on the same medium solidified with agar (P1 >> P2). The growth phase of the bacteria did not have any effect on the tRNA(trp) elution profile. Changes in tRNA(trp) induced by HTF were readily and completely reversible when the cells were transferred to an iron-rich medium, but those induced by desferrioxamine remained irreversible for a long period (16 h) after such transfer.
Culture Media↗
Two essential regions for tRNA recognition in Bacillus subtilis tryptophanyl-tRNA synthetase.
Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) is a homodimeric enzyme. A model for its ability to recognize tRNA(Trp) in B. subtilis was proposed by using computer modelling. This was based on the the fact that there is high homology among bacterial TrpRSs [Chen, Jiang, Jin and Wang (2001) Acta Biochim. Biophys. Sinica 33, 687-690], in which the enzyme dimer binds to two tRNA(Trp) molecules and each tRNA(Trp) is bound to two different domains across the surface of the dimer. In this work, three deletion mutants of TrpRS were constructed and their products were purified. After determining the kinetic parameters of the mutants in the two-step reaction, it was found that the relative activities of wild-type and mutant enzymes had changed little in the ATP-pyrophosphate exchange reaction. In contrast, the activities of three mutant proteins were much decreased in the tRNA(Trp) aminoacylation assay. Deletion of residues 108-122 and residues 234-238 caused 44% and 80% reductions in the activity, respectively. When both regions were deleted, the aminoacylation activity of the TrpRS mutant was too low to be determined using tRNA(Trp) at the limiting concentration. Gel-retardation assays showed that the acceptor minihelix and the anticodon microhelix were recognized by the domains of TrpRS spanning residues 108-122 and residues 234-238 respectively. In addition, the deletion of amino acids 234-238 affected the normal induced expression of TrpRS at 37 degrees C. In conclusion, residues 108-122 and 234-238 were found essential for tRNA(Trp) recognition.
Adenosine Triphosphate↗
Heterogeneities in vertebrate tRNAs(Trp) avian retroviruses package only as a primer the tRNA(Trp) lacking modified m2G in position 7.
Bovine, rabbit and chicken tRNA(Trp) species and tRNA(Trp) packaged in avian myeloblastosis virus were separated and purified using two-dimensional gel electrophoresis and their primary structures were determined. Two major tRNA(Trp) species (1 and 2) were identified in beef and rabbit, two minor ones (3 and 4) in beef and only one minor in rabbit. Their structures differ by 4 nucleotide substitutions located in the D, S and T loops (positions 16, 47, 57 and 59). Species 3 and 4 differ from one another by only one nucleotide at position 2. Differences between tRNA(Trp) species were also observed in the extent of methylation of some nucleotides. Chicken tRNA(Trp) presents only one species similar to the mammalian type 1 tRNA(Trp). In the case of the three studies animals this tRNA could be separated into two subspecies, which differ by a post-transcriptional modification of nucleotide 7 in the acceptor stem: G or m2G. However only the nonmethylated species is used as the primer of DNA-RNA directed retrotranscription since it is only that form which was found in avian retroviruses. The methylation of G to m2G at position 7 could thus prevent the recognition of tRNA(Trp) by retroviral protein(s) responsible for the selective packaging of the primer tRNA(Trp).
Animals↗
3'-immature tRNA(Trp) is required for ribosome inactivation by gelonin,a plant RNA N-glycosidase.
Inactivation of ribosomes by gelonin, a ribosome-inactivating protein with RNA N-glycosidase activity on 28 S rRNA, requires macromolecular cofactors present in post-ribosomal supernatants. One of these cofactors has been purified from a rat liver extract and identified as an RNA about 70 nt long which in sequence analysis shows a high level of similarity with mammalian (bovine) tRNA(Trp). The pattern of the sequencing gel is consistent with the co-existence in the preparation of two 3'-immature tRNA(Trp) species, missing only A75, or both A75 and C74. In the presence of ATP, CTP and tRNA nucleotidyltransferase, the gelonin-stimulating RNA is a good acceptor of tryptophan. An oligodeoxynucleotide complementary to positions 55 to 72 of mammalian (bovine) tRNA(Trp) hybridizes with the gelonin-stimulating RNA as demonstrated by gel mobility shift and ribonuclease H digestion. The oligodeoxynucleotide-directed ribonuclease H treatment also abolishes the gelonin-promoting activity of crude preparations of RNA, giving strong evidence that the only active RNA is a tRNA(Trp)-like molecule.
Animals↗