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PubMed1992

Chloroplast heteroplasmicity is stabilized by an amber-suppressor tryptophan tRNA(CUA).

Photosynthesis-deficient mutants of the green alga Chlamydomonas reinhardtii were previously shown to arise from nonsense mutations within the chloroplast rbcL gene, which encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Photosynthesis-competent revertants of these nonsense mutants have often been found to be stably heteroplasmic, giving rise to both mutant and revertant cells during mitotic or meiotic divisions under nonselective growth conditions. It was proposed that the stable heteroplasmicity might arise from a balanced polymorphism of suppressor and wild-type alleles of a tRNA gene maintained within the polyploid chloroplast genome. In the present study, we have focused on determining the molecular basis for the heteroplasmicity of one such revertant, named R13-3C, which was recovered from the 18-7G amber (UAG) mutant. Restriction-enzyme analysis and DNA sequencing showed that the amber mutation is still present in the rbcL gene of the revertant strain. In contrast, DNA sequencing of the suspected tRNA(Trp) gene of the revertant revealed a mutation that would change its CCA anticodon to amber-specific CUA. This mutation was found to be heteroplasmic, being present in only 70% of the tRNA(Trp) gene copies. Under nonselective conditions, the suppressor mutation was lost from cells that also lost the revertant phenotype. We conclude that stable heteroplasmicity can arise as a balanced polymorphism of organellar alleles. This observation suggests that additional tRNA suppressors may be identified due to their heteroplasmic nature within polyploid genomes.

Animals↗

PubMedDate not supplied

A mouse tRNA pseudogene derived from a tRNA(Trp) coding sequence.

The identification and characterization of a mouse tRNA(Trp) pseudogene is reported. A synthetic oligonucleotide (31 mer), identical with the 3' half of a tRNA(Trp), was used to examine three mouse lambda clones that are known to contain clusters of tRNA genes. A fragment of one of these lambda clones strongly hybridizes to the oligonucleotide; the sequence of this region shows the presence of a gene significantly homologous to the chick tRNA(Trp). However two point mutations and a three base deletion prevent the folding of a possible transcript of this gene. The presence of a conserved promoter sequence for RNA polymerase III, that should allow the transcription of this gene, does not ensure the transcription of the gene, at least in our in vitro system.

Animals↗

PubMed2004

Sequential 2'-O-methylation of archaeal pre-tRNATrp nucleotides is guided by the intron-encoded but trans-acting box C/D ribonucleoprotein of pre-tRNA.

Haloferax volcanii pre-tRNA(Trp) processing requires box C/D ribonucleoprotein (RNP)-guided 2'-O-methylation of nucleotides C34 and U39 followed by intron excision. Positioning of the box C/D guide RNA within the intron of this pre-tRNA led to the assumption that nucleotide methylation is guided by the cis-positioned box C/D RNPs. We have now investigated the mechanism of 2'-O-methylation for the H. volcanii pre-tRNA(Trp) in vitro by assembling methylation-competent box C/D RNPs on both the pre-tRNA and the excised intron (both linear and circular forms) using Methanocaldococcus jannaschii box C/D RNP core proteins. With both kinetic studies and single nucleotide substitutions of target and guide nucleotides, we now demonstrate that pre-tRNA methylation is guided in trans by the intron-encoded box C/D RNPs positioned in either another pre-tRNA(Trp) or in the excised intron. Methylation by in vitro assembled RNPs prefers but does not absolutely require Watson-Crick pairing between the guide and target nucleotides. We also demonstrate for the first time that methylation of two nucleotides guided by a single box C/D RNA is sequential, that is, box C'/D' RNP-guided U39 methylation first requires box C/D RNP-guided methylation of C34. Methylation of the two nucleotides of exogenous pre-tRNA(Trp) added to an H. volcanii cell extract also occurs sequentially and is also accomplished in trans using RNPs that pre-exist in the extract. Thus, this trans mechanism is analogous to eukaryal pre-rRNA 2'-O-methylation guided by intron-encoded but trans-acting box C/D small nucleolar RNPs. This trans mechanism could explain the observed accumulation of the excised H. volcanii pre-tRNA(Trp) intron in vivo. A trans mechanism would also eliminate the obligatory refolding of the pre-tRNA that would be required to carry out two cis-methylation reactions before pre-tRNA splicing.

Archaeal Proteins↗

PubMed1989

Tryptophanyl-tRNA synthetase from Bacillus subtilis. Characterization and role of hydrophobicity in substrate recognition.

The tryptophanyl-tRNA synthetase from Bacillus subtilis was purified to homogeneity and characterized. It has an alpha 2 subunit structure and a molecular weight of 77,000. Tryptophanyl-tRNA synthetase does not catalyze any significant proofreading. It activates tryptophan as well as the three fluorinated analogues, DL-4-fluoro-, DL-5-fluoro-, or DL-6-fluorotryptophan (4F-, 5F-, and 6F-Trp), in the ATP-pyrophosphate exchange reaction. In the aminoacylation reaction, the fluorotryptophans act as competitive inhibitors of Trp. Their relative activities follow the same order in both reactions: Trp greater than 4F-Trp greater than 6F-Trp greater than 5F-Trp. This order is the inverse of the order of relative hydrophobicities of these compounds, pointing to the importance of hydrophobic interactions in the selective recognition by tryptophanyl-tRNA synthetase among this group of substrates. To define the physical basis of the relative hydrophobicities, the crystallographic structure of 4F-Trp was determined and compared to that of trptophan. Charge distributions calculated for tryptophan and its different fluoroanalogues on the basis of molecular structures were supported by their carbon-13 NMR spectra. Correlations between charge distributions and relative hydrophobicities suggest that the polarity of the C-F bond represents an underlying factor determining the hydrophobicities of 4F-, 5F-, and 6F-Trp, thus relating tryptophanyl-tRNA synthetase selectivity toward tryptophan and its fluoroanalogues directly to their electronic configurations.

Adenosine Monophosphate↗

PubMed1994

Changes in translational accuracy of Escherichia coli when folate metabolism is perturbed.

Translational accuracy was monitored in Escherichia coli mutants which contain abnormal folate pools. A decrease in translational accuracy was indicated by the increased production of a T4 phage mutant containing a UGA mutation in a tail fibre gene. An E. coli folC mutant suppressed the phage mutant under conditions where it presumably accumulated methyl-tetrahydrofolate (methyl-THF). A UGA suppressor strain with the mutation affecting the primary structure of the tRNA(trp) normally suppressed the phage mutant. When the strain was made thymine-requiring it no longer suppressed. The accumulation of methyl-THF which permits suppression by thymine-requiring strains may act to interfere with suppression by the tRNA suppressor, possibly by changing the modification pattern of the tRNA.

Aminopterin↗

PubMed1995

A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity.

We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn is used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methylpurine ribonucleoside. Of the reaction products, ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and hence provides a spectrophotometric signal that can be continuously followed. The non-destructive nature of the spectrophotometric assay allowed the re-use of the tRNAs in question in successive experiments. The usefulness of this method was demonstrated for glutaminyl-tRNA synthetase (GlnRS) and tryptophanyl-tRNA synthetase. Initial velocities measured using this assay correlate closely with those assayed by quantitation of [3H]Gln-tRNA or [14C]Trp-tRNA formation respectively. In both cases amino acid transfer from the aminoacyl adenylate to the tRNA represents the rate determining step. In addition, aminoacyl adenylate formation by aspartyl-tRNA synthetase was followed and provided a more sensitive means of active site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GlnRS.

Acylation↗

PubMed1993

Inhibition of expression of the tryptophanase operon in Escherichia coli by extrachromosomal copies of the tna leader region.

Expression of the tryptophanase (tna) operon in Escherichia coli is regulated by catabolite repression and transcription attenuation. Expression is induced by the presence of elevated levels of tryptophan in a growth medium devoid of a catabolite-repressing carbon source. Induction requires the translation of a 24-residue coding region, tnaC, located in the 319-nucleotide transcribed leader region preceding tnaA, the structural gene for tryptophanase. Multicopy plasmids carrying the tnaC leader region were found to inhibit induction of the chromosomal tna operon. Mutational studies established that this inhibition was not due to inhibited transcription initiation, translation initiation, tryptophan transport, or enzyme activity. Rather, multicopy tnaC plasmids inhibited induction by preventing tryptophan-induced transcription antitermination in the leader region of the tna operon. Translation of the single Trp codon in tnaC of the multicopy plasmids was shown to be essential for this inhibition. We hypothesize that translation of the Trp codon of the leader peptide titrates out a trans-acting factor that is essential for tryptophan-induced antitermination in the chromosomal tna operon. We postulate that this factor is an altered form of tRNATrp.

Alleles↗

PubMed2002

Three G.C base pairs required for the efficient aminoacylation of tRNATrp by tryptophanyl-tRNA synthetase from Bacillus subtilis.

Acceptor stem is an essential region in the recognition of tRNAs by their cognate aminoacyl-tRNA synthetase. In this study, a library containing 20 nt random region and tryptophanyl-tRNA synthetase (TrpRS) from Bacillus subtilis were used for in vitro selection to find a new structural feature in the tRNA(Trp) acceptor stem sequence that is required for B. subtilis TrpRS recognition. After three rounds of selection, the TrpRS binding RNAs dominate the RNA pool. The aptamers share a common structure of three G.C base pairs, which was also found in the acceptor stem of wild-type B. subtilis tRNA(Trp). A series of tRNA(Trp) variants was prepared by in vitro transcription, and their efficiencies of tryptophanylation (k(cat)/K(M)) were measured with the aid of TrpRS from B. subtilis. The mutants that possess the three G.C base pairs and G73 discriminator base exhibit almost the same aminoacylation efficiencies as B. subtilis tRNA(Trp), while the G73 discriminator base itself cannot confer efficient aminoacylation to the tRNA(Trp) molecule. Thus, these three base pairs (G2.C71, G3.C70, and G4.C69) in the B. subtilis tRNA(Trp) acceptor stem were established to be new identity elements, and their importance was between the previously characterized major element G73 and minor elements A1/U72 and G5/C68. The minimum set of identity elements that is required to confer efficient aminoacylation by B. subtilis TrpRS included G73, G2.C71, G3.C70, and G4.C69.

Acylation↗

PubMed2002

Recognition by tryptophanyl-tRNA synthetases of discriminator base on tRNATrp from three biological domains.

To study the recognition by tryptophanyl-tRNA synthetase (TrpRS) of tRNA(Trp) discriminator base, mutations were introduced into the discriminator base of Bacillus subtilis, Archeoglobus fulgidus, and bovine tRNA(Trp), representing the three biological domains. When B. subtilis, A. fulgidus, and human TrpRS were used to acylate these tRNA(Trp), two distinct preference profiles regarding the discriminator base of different tRNA(Trp) substrates were found: G>A>U>C for B. subtilis TrpRS, and A>C>U>G for A. fulgidus and human TrpRS. The preference for G73 in tRNA(Trp) by bacterial TrpRS is much stronger than the modest preferences for A73 by the archaeal and eukaryotic TrpRS. Cross-species reactivities between TrpRS and tRNA(Trp) from the three domains were in accordance with the view that the evolutionary position of archaea is intermediate between those of eukarya and bacteria. NMR spectroscopy revealed that mutation of A73 to G73 in bovine tRNA(Trp) elicited a conformational alteration in the G1-C72 base pair. Mutation of G1-C72 to A1-U72 or disruption of the G1-C72 base pair also caused reduction of Trp-tRNA(Trp) formation. These observations identify a tRNA(Trp) structural region near the end of acceptor stem comprising A73 and G1-C72 as a crucial domain required for effective recognition by human TrpRS.

Animals↗

PubMed1988

A tRNA(Trp) intron endonuclease from Halobacterium volcanii. Unique substrate recognition properties.

We report on the properties of a partially purified tRNA intron endonuclease from the archaebacterium Halobacterium volcanii. This enzyme is capable of precise excision of the 104-nucleotide intron from halo-bacterial pre-tRNA(Trp) substrates generated in vitro by T7 RNA polymerase transcription. The reaction requires divalent cations (Mg2+ or Ca2+) or spermidine, is inhibited by monovalent cations, and produces 5'-hydroxyl and 2',3'-cyclic phosphate termini. Unlike the universal substrate recognition properties characteristic of the eukaryotic tRNA intron endonucleases, this enzyme is specific for halophilic tRNA(Trp) substrates. The partially purified enzyme is not capable of removing the intron from a yeast pre-tRNA(Phe) substrate. Analysis of the enzyme's ability to cleave tRNA(Trp) substrates lacking exon sequences demonstrated that the mature tRNA-like structure is not required in the substrate. A substrate retaining the intact intron and only the anticodon stem and loop exon regions was efficiently cleaved. Deletions within the intron indicated that the intron was not a primary site for recognition by the endonuclease; however, its presence affects the efficiency of the cleavage reaction. The possible relationship of this enzyme to other RNA endonucleases is discussed.

Base Sequence↗

PubMed1998

The mitochondrial genome of the sea anemone Metridium senile (Cnidaria): introns, a paucity of tRNA genes, and a near-standard genetic code.

The circular, 17,443 nucleotide-pair mitochondrial (mt) DNA molecule of the sea anemone, Metridium senile (class Anthozoa, phylum Cnidaria) is presented. This molecule contains genes for 13 energy pathway proteins and two ribosomal (r) RNAs but, relative to other metazoan mtDNAs, has two unique features: only two transfer RNAs (tRNA(f-Met) and tRNA(Trp)) are encoded, and the cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes each include a group I intron. The COI intron encodes a putative homing endonuclease, and the ND5 intron contains the molecule's ND1 and ND3 genes. Most of the unusual characteristics of other metazoan mtDNAs are not found in M. senile mtDNA: unorthodox translation initiation codons and partial translation termination codons are absent, the use of TGA to specify tryptophan is the only genetic code modification, and both encoded tRNAs have primary and secondary structures closely resembling those of standard tRNAs. Also, with regard to size and secondary structure potential, the mt-s-rRNA and mt-1-rRNA have the least deviation from Escherichia coli 16S and 23S rRNAs of all known metazoan mt-rRNAs. These observations indicate that most of the genetic variations previously reported in metazoan mtDNAs developed after Cnidaria diverged from the common ancestral line of all other Metazoa.

Amino Acid Sequence↗

PubMed2006

Structure of human tryptophanyl-tRNA synthetase in complex with tRNATrp reveals the molecular basis of tRNA recognition and specificity.

Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes responsible for the covalent link of amino acids to their cognate tRNAs. The selectivity and species-specificity in the recognitions of both amino acid and tRNA by aaRSs play a vital role in maintaining the fidelity of protein synthesis. We report here the first crystal structure of human tryptophanyl-tRNA synthetase (hTrpRS) in complex with tRNA(Trp) and Trp which, together with biochemical data, reveals the molecular basis of a novel tRNA binding and recognition mechanism. hTrpRS recognizes the tRNA acceptor arm from the major groove; however, the 3' end CCA of the tRNA makes a sharp turn to bind at the active site with a deformed conformation. The discriminator base A73 is specifically recognized by an alpha-helix of the unique N-terminal domain and the anticodon loop by an alpha-helix insertion of the C-terminal domain. The N-terminal domain appears to be involved in Trp activation, but not essential for tRNA binding and acylation. Structural and sequence comparisons suggest that this novel tRNA binding and recognition mechanism is very likely shared by other archaeal and eukaryotic TrpRSs, but not by bacterial TrpRSs. Our findings provide insights into the molecular basis of tRNA specificity and species-specificity.

Amino Acid Sequence↗

PubMed1990

5-[[(carboxymethyl)amino]methyl]uridine is found in the anticodon of yeast mitochondrial tRNAs recognizing two-codon families ending in a purine.

The modified nucleoside (U*) present in the wobble position of Saccharomyces cerevisiae mitochondrial tRNA(Leu) and tRNA(Trp) was isolated by thin-layer chromatography and HPLC. Its chromatographic, UV spectral, and mass spectrometric properties were shown to be identical with those of 5- [[(carboxymethyl)amino]methyl]uridine (cmnm5U). This nucleoside found in yeast mitochondrial tRNAs reading two-codon families ending in a purine permits the selective recognition of A and G in the third codon position.

Anticodon↗

PubMed1993

Identity of Saccharomyces cerevisiae tRNA(Trp) is not changed by an anticodon mutation that creates an amber suppressor.

A C35-->T mutation in an Escherichia coli tRNA(Trp) gene creates an amber suppressor which efficiently inserts glutamine in response to UAG codons in vivo (Soll, L., and Berg, P. (1969) Nature 223, 1340-1342). We have introduced the same change in a yeast tRNA(Trp) gene and demonstrated that the tRNA acts as an efficient amber suppressor in vivo. Amino acid sequence analyses were performed on chitinase produced by cells carrying the corresponding gene with a UAG codon at position 8 of the mature protein plus the mutant tRNA(Trp) gene. In contrast to comparable experiments with E. coli, tryptophan is inserted at a frequency > or = 80% by the yeast suppressor tRNA(Trp). Furthermore, in vitro charging experiments with the mutant tRNA(Trp) reveal no detectable increase in glutamine acceptor activity results from the C35-->T transition. The identity elements in E. coli tRNA(Gln) are well characterized (Jahn, M., Rogers, J., and Soll, D. (1991) Nature 352, 258-260). Sequence comparisons of the tRNA(Trp) and tRNA(Gln) molecules from E. coli reveal that the amber suppressor tRNA(Trp) has four of five identity elements required for glutaminyl-tRNA synthetase recognition. A similar comparison in the yeast system shows only two of the five potential identity elements are present. We conclude that, in spite of substantial structural similarities between yeast and E. coli aminoacyl-tRNA synthetases, fundamental differences can exist with regard to tRNA recognition.

Amino Acyl-tRNA Synthetases↗

PubMed1996

Influence of the last amino acid in the nascent peptide on EF-Tu during decoding.

The last two amino acids of the nascent peptide at the ribosomal P-site influence the efficiency of termination readthrough at the stop codon UGA (Mottagui-Tabar et al (1994) EMBO J 13, 249-257; Björnsson et al (1996) EMBO J 15, 1696-1704). Here we analyze this effect on readthrough by wild type or a UGA suppressor form (Su9) of tRNA(Trp) by varying the codons at positions-1 and -2 at the 5' side of UGA. Strains with wild-type or mutant (ArBr) forms of elongation factor Tu (EF-Tu) were analyzed (Vijgenboom et al (1985) EMBO J4, 1049-1052). The effect on readthrough by changing these-1 and -2 codons is different on the two forms of tRNA(Trp) and is also dependent on the structure of EF-Tu. Readthrough by the tRNA(Trp)-derived suppressor, but not wild-type tRNA(Trp), is sensitive to the van der Waals volume of the last amino acid in the nascent peptide. Together with mutant EF-Tu, both forms of tRNA(Trp) are sensitive. The data suggest that the C-terminal amino acid in the nascent peptide is in a functional interaction with the EF-Tu ternary complex. This interaction is changed by mutation in tRNA(Trp) at position 24 or in EF-Tu at position 375. No indication of a changed interaction between the mutant EF-Tu and the penultimate amino acid could be found. Mutant forms of RF2 (Mikuni et al (1991) Biochimie 73, 1509-1516) and ribosomal proteins S4 and S12 (Fáxen et al (1988) J Bacteriol 170, 3756-3760) were found not be altered in sensitivity to the last two amino acids in the nascent peptide.

Codon↗

PubMed1992

Conversion of a methionine initiator tRNA into a tryptophan-inserting elongator tRNA in vivo.

The role of the anticodon and discriminator base in aminoacylation of tRNAs with tryptophan has been explored using a recently developed in vivo assay based on initiation of protein synthesis by mischarged mutants of the Escherichia coli initiator tRNA. Substitution of the methionine anticodon CAU with the tryptophan anticodon CCA caused tRNA(fMet) to be aminoacylated with both methionine and tryptophan in vivo, as determined by analysis of the amino acids inserted by the mutant tRNA at the translational start site of a reporter protein containing a tryptophan initiation codon. Conversion of the discriminator base of tRNA(CCA)fMet from A73 to G73, the base present in tRNA(Trp), eliminated the in vivo methionine acceptor activity of the tRNA and resulted in complete charging with tryptophan. Single base changes in the anticodon of tRNA(CCA)fMet containing G73 from CCA to UCA, GCA, CAA, and CCG (changes underlined) essentially abolished tryptophan insertion, showing that all three anticodon bases specify the tryptophan identity of the tRNA. The important role of G73 in tryptophan identity was confirmed using mutants of an opal suppressor derivative of tRNA(Trp). Substitution of G73 with A73, C73, or U73 resulted in a large loss of the ability of the tRNA to suppress an opal stop codon in a reporter protein. Base pair substitutions at the first three positions of the acceptor stem of the suppressor tRNA caused 2-12-fold reductions in the efficiency of suppression without loss of specificity for aminoacylation of the tRNA with tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticodon↗

PubMed1990

Construction of an opal suppressor by oligonucleotide-directed mutagenesis of a Saccharomyces cerevisiae tRNA(Trp) gene.

In vitro mutagenesis was used to create putative opal suppressor alleles of a tRNA(Trp) gene of Saccharomyces cerevisiae. The construct with the requisite anticodon change did not result in an active suppressor, whereas when a second change was introduced into the portion of the gene encoding the intron, an active and specific opal suppressor was produced. We propose that the secondary structure of transcripts from the first mutant may prevent efficient pre-tRNA processing, whereas normal processing occurs with the double mutant.

Alleles↗

PubMed1995

Analysis of the role of 5' and 3' flanking sequence elements upon in vivo expression of the plant tRNATrp genes.

We have isolated the majority (seven) of the tRNA(Trp) genes of Arabidopsis and have studied the 5' and 3' flanking sequence requirements for their efficient expression in vivo by using an assay requiring translational suppression of the luciferase reporter gene. The expressed tRNA(Trp) genes contain no highly conserved 5' flanking sequences; however, these sequences are distinctly AT rich, contain several possible TATA elements, and are bound in vitro by recombinant plant TATA binding protein. Replacement of the natural 5' flanking sequences with three different sequences lacking TATA elements reduced expression in vivo up to 10-fold; the same effect was observed when the TATA elements of the natural 5' sequences were inactivated by point mutations. Introduction of a single TATA element from the adenovirus major late promoter into an artificial 5' flanking region of the tRNA(Trp) gene enhanced expression in vivo when the TATA element was placed at position -32 relative to the first nucleotide of the mature tRNA sequence, but not when it was placed at position -24. Primer extension analyses of in vitro transcripts revealed that the position of the TATA element helps dictate the start site of transcription. Efficient expression of the tRNA genes in vivo also required 3' flanking sequences capable of terminating transcription.

Arabidopsis↗