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PubMed2004

An unusual tryptophanyl tRNA synthetase interacts with nitric oxide synthase in Deinococcus radiodurans.

In mammals, nitric oxide synthases (NOSs) produce nitric oxide for signaling and defense functions; in Streptomyces, NOS proteins nitrate a tryptophanyl moiety in synthesis of a phytotoxin. We have discovered that the NOS protein from the radiation-resistant bacterium Deinococcus radiodurans (deiNOS) associates with an unusual tryptophanyl tRNA synthetase (TrpRS). D. radiodurans contains genes for two TrpRSs: the first has approximately 40% sequence identity to typical TrpRSs, whereas the second, identified as the NOS-interacting protein (TrpRS II), has only approximately 29% identity. TrpRS II is induced after radiation damage and contains an N-terminal extension similar to those of proteins involved in stress responses. Recombinantly expressed TrpRS II binds tryptophan (Trp), ATP, and D. radiodurans tRNA(Trp) and catalyzes the formation of 5' adenyl-Trp and tRNA(Trp), with approximately five times less activity than TrpRS I. Upon coexpression in Escherichia coli, TrpRS II binds to, copurifies with, and dramatically enhances the solubility of deiNOS. Dimeric TrpRS II binds dimeric deiNOS with a stoichiometry of 1:1 and a dissociation constant of 6-30 muM. Upon forming a complex, deiNOS quenches the fluorescence of an ATP analog bound to TrpRS II, and increases its affinity for substrate l-arginine. Remarkably, TrpRS II also activates the NOS activity of deiNOS. These findings reveal a link between bacterial NOS and Trp metabolism in a second organism and may indicate yet another novel biological function for bacterial NOS.

Adenosine Triphosphate↗

PubMed1990

Relationship between protein synthesis and concentrations of charged and uncharged tRNATrp in Escherichia coli.

We have continuously monitored Trp-tRNA(Trp) concentrations in vivo and, in the same cultures, measured rates of protein synthesis in isogenic stringent and relaxed strains. We have also manipulated cellular charged and uncharged [tRNA(Trp)] by two means: (i) the strain used contains a Trp-tRNA synthetase mutation that increases the Km for Trp; thus, varying exogenous Trp varies cellular Trp-tRNA(Trp); and (ii) we have introduced into the mutant strain a plasmid containing the tRNA(Trp) gene behind an inducible promoter; thus, total [tRNA(Trp)] also can be varied depending on length of induction. The use of these conditions, combined with a previously characterized assay system, has allowed us to demonstrate that (i) the rate of incorporation of Trp into protein is proportional to the fraction of tRNA(Trp) that is charged; for any given total [tRNA(Trp)], this rate is also proportional to the [Trp-tRNA(Trp)]; (ii) uncharged tRNA(Trp) inhibits incorporation of Trp into protein; and (iii) rates of incorporation into protein of at least two other amino acids, Lys and Cys, are also sensitive to [Trp-tRNA(Trp)] and are inhibited by uncharged tRNA(Trp). Our results are consistent with models of translational control that postulate modulating polypeptide chain elongation efficiency by varying concentrations of specific tRNAs.

Amino Acyl-tRNA Synthetases↗

PubMed1993

Anticodon bases C34 and C35 are major, positive, identity elements in Saccharomyces cerevisiae tRNA(Trp).

A single form of tRNA(Trp) exists in the yeast cytoplasm to respond to the unique codon, UGG, which specifies this amino acid. Mutations in the anticodon of the corresponding gene, which generate potential nonsense suppressor tRNAs, have been generated in vitro and tested in vivo for biological activity. The amber (C35U) and opal (C34U) suppressors show strong and weak activities respectively while the ochre suppressor (C34U,C35U) has no detectable biological activity. To understand the basis for these differences, a set of synthetic tRNA(Trp) genes has been constructed to permit in vitro, T7 RNA polymerase synthesis of transcripts corresponding to the normal and mutant tRNAs. Kinetic parameters for aminoacylation of these transcripts by purified, yeast, tryptophanyl-tRNA synthetase have been measured and compared to values observed using the naturally occurring tRNA(Trp) as a substrate. The efficiency of aminoacylation is reduced by 40, 2000, and 30,000 fold by the C35U, C34U, and C34U,C35U mutations respectively. Interestingly, the C35U change affects only tRNA binding while C34U also alters catalytic efficiency. We conclude that both C34 and C35 are major identity elements in the recognition of tRNA(Trp) by its cognate synthetase. These differences in aminoacylation efficiency closely parallel the in vivo suppressor activities of the mutants.

Acylation↗

PubMed1995

Evidence that uncharged tRNA can inhibit a programmed translational frameshift in Escherichia coli.

In the modified release factor 2 (RF2) programmed translational frameshift (with a sense codon replacing the wild-type in-frame UGA codon at the shift site), ribosomes shift +1 into the reading frame for an out-of-frame reporter fused to the frameshift sequence. Partitioning of ribosomes between the out-of-frame shift and in-frame reading depends on the codon at the shift site and on the levels of tRNA decoding the in-frame codon. Overexpression of a tRNA species cognate to the in-frame codon at the shift site significantly reduces the frequency of frame-shifting, presumably by facilitating in-frame reading, which would reduce production of the out-of-frame reporter. However, since overexpression of a tRNA increases levels of both charged and uncharged tRNA, it is possible that uncharged cognate tRNA might be able to reduce the frequency of the frameshift, by entering the A site on the ribosome. To test this, we manipulated charged and uncharged tRNA levels in vivo, using the tryptophan analog tryptophan hydroxamate, which increases the proportion of uncharged tRNA(Trp) by competing with cognate amino acid tryptophan for tryptophanyl-tRNA synthetase, thereby reducing protein synthesis. We report here that a slight but reproducible reduction in the relative frequency of the frameshift is observed when tryptophan hydroxamate is added to cells containing the modified RF2 shift with UGG (Trp codon) at the shift site. When tRNA(Trp) is overexpressed from another plasmid, the shift frequency drops three- to fourfold, as expected, however, this reduction is still seen in the presence of the analog. Thus, under conditions when most of the tRNA(Trp) is apparently uncharged, excess tRNA(Trp) still causes a significant reduction in the frameshift when UGG is at the shift site, providing evidence that uncharged cognate tRNA also can inhibit this frameshift.

Amino Acyl-tRNA Synthetases↗

PubMed2002

NMR analysis of bovine tRNATrp: conformation dependence of Mg2+ binding.

NMR was used to study the solution structure of bovine tRNA(Trp) hyperexpressed in Escherichia coli. With the use of (15)N labeling and site-directed mutagenesis to assign overlapping resonances through the base pair replacement of U(71)A(2) by G(2)C(71), U(27)A(43) by G(27)C(43), and G(12)C(23) by U(12)A(23), the resonances of all 26 observable imino protons in the helical regions and in the tertiary interactions were assigned unambiguously by means of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear single quantum coherence methods. When the discriminator base A(73) and the G(12)C(23) base pair on the D stem, two identity elements on bovine tRNA(Trp) that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G(73) and U(12)A(23), respectively, NMR analysis revealed an important conformational change in the U(12)A(23) mutant but not in the G(73) mutant molecule. Thus A(73) appears to be directly recognized by tryptophanyl-tRNA synthetase, and G(12)C(23) represents an important structural determinant. Mg(2+) effects on the assigned resonances of imino protons allowed the identification of strong, medium, and weak Mg(2+) binding sites in tRNA(Trp). Strong Mg(2+) binding modes were associated with the residues G(7), s(4)U(8) (where s(4)U is 4-thiouridine), G(12), and U(52). The observations that G(42) was associated with strong Mg(2+) binding in only the U(12)A(23) mutant tRNA(Trp) but not the wild type or G(73) mutant tRNA(Trp) and that the G(7), s(4)U(8), G(24), and G(22) imino protons are associated with a two-site Mg(2+) binding mode in wild type and G(73) mutant but only a one-site mode in the U(12)A(23) mutant established the occurrence of conformational change in the U(12)A(23) mutant tRNA(Trp). These observations also established the dependence of Mg(2+) binding on tRNA conformation and the usefulness of Mg(2+) binding sites as conformational probes. The thermal titration of tRNA(Trp) in the presence and absence of 10 mm Mg(2+) indicated that overall tRNA(Trp) structure stability was increased by more than 15 degrees C by the presence of Mg(2+).

Animals↗

PubMed2001

Nucleotides U28-A42 and A37 in unmodified yeast tRNA(Trp) as negative identity elements for bovine tryptophanyl-tRNA synthetase.

Wild-type bovine and yeast tRNA(Trp) are efficiently aminoacylated by tryptophanyl-tRNA synthetase both from beef and from yeast. Upon loss of modified bases in the synthetic transcripts, mammalian tRNA(Trp) retains the double recognition by the two synthetases, while yeast tRNA(Trp) loses its substrate properties for the bovine enzyme and is recognised only by the cognate synthetase. By testing chimeric bovine-yeast transcripts with tryptophanyl-tRNA synthetase purified from beef pancreas, the nucleotides responsible for the loss of charging of the synthetic yeast transcript have been localised in the anticodon arm. A complete loss of charging akin to that observed with the yeast transcript requires substitution in the bovine backbone of G37 in the anticodon loop with yeast A37 and of C28-G42 in the anticodon stem with yeast U28-A42. Since A37 does not prevent aminoacylation of the wild-type yeast tRNA(Trp) by the beef enzyme, a negative combination apparently emerges in the synthetic transcript after unmasking of U28 by loss of pseudourydilation.

Adenine↗

PubMed1993

Modified nucleotides in Bacillus subtilis tRNA(Trp) hyperexpressed in Escherichia coli.

In the present study, modified nucleotides in the B. subtilis tRNA(Trp) cloned and hyperexpressed in E. coli have been identified by TLC and HPLC analyses. The modification patterns of the two isoacceptors of cloned B. subtilis tRNA(Trp) have been compared with those of native tRNA(Trp) from B. subtilis and from E. coli. The modifications of the A73 mutant of B. subtilis tRNA(Trp), which is inactive toward its cognate TrpRS, were also investigated. The results indicate the formation of the modified nucleotides S4U8, Gm18, D20, Cm32, i6A/ms2i6A37, T54 and psi 55 on cloned B. subtilis tRNA(Trp). This modification pattern resembles the pattern of E. coli tRNA(Trp), except that m7G is missing from the cloned tRNA(Trp), probably on account of its short extra loop. In contrast, the pattern departs substantially from that of native B. subtilis tRNA(Trp). Therefore, the cloned B. subtilis tRNA(Trp) has taken on largely the modification pattern of E. coli tRNA(Trp) despite the 26% sequence difference between the two species of tRNA, gaining in particular the Cm32 and Gm18 modifications from the E. coli host. A notable difference between the isoacceptors of the cloned tRNA(Trp) was seen in the extent of modification of A37, which occurred as either the hypomodified i6A or the hypermodified ms2i6A form. Surprisingly, base substitution of guanosine by adenosine at position 73 of the cloned tRNA(Trp) has led to the abolition of the 2'-O-methylation modification of the remote G18 residue.

Bacillus subtilis↗

PubMed1998

Identity elements and aminoacylation of plant tRNATrp.

Mutation of the Arabidopsis thaliana tRNA (Trp)(CCA) anticodon or of the A73 discriminator base greatly diminishes in vitro aminoacylation with tryptophan, indicating the importance of these nucleotides for recognition by the plant tryptophanyl-tRNA synthetase. Mutation of the tRNA (Trp)(CCA) anticodon to CUA so as to translate amber nonsense codons permits tRNA (Trp)(CCA) to be aminoacylated by A.thaliana lysyl-tRNA synthetase. Thus, translational suppression by tRNA (TRP)(CCA) observed in plant cells includes significant incorporation of lysine into protein.

Animals↗

PubMed2002

Modification of the universally unmodified uridine-33 in a mitochondria-imported edited tRNA and the role of the anticodon arm structure on editing efficiency.

Editing of tRNA has a wide phylogenetic distribution among eukaryotes and in some cases serves to expand the decoding capacity of the target tRNA. We previously described C-to-U editing of the wobble position of the imported tRNA(Trp) in Leishmania mitochondria, which is essential for decoding UGA codons as tryptophan. Here we show the complete set of nucleotide modifications in the anticodon arm of the mitochondrial and cytosolic tRNA(Trp) as determined by electrospray ionization mass spectrometry. This analysis revealed extensive mitochondria-specific posttranscriptional modifications, including the first example of thiolation of U33, the "universally unmodified" uridine. In light of the known rigidity imparted on sugar conformation by thiolation, our discovery of a thiolated U33 suggests that conformational flexibility is not a universal feature of the anticodon structural signature. In addition, the in vivo analysis of tRNA(Trp) variants presented shows a single base-pair reversal in the anticodon stem of tRNA(Trp) is sufficient to abrogate editing in vivo, indicating that subtle changes in anticodon structure can have drastic effects on editing efficiency.

Anticodon↗

PubMed1993

The functional analysis of nonsense suppressors derived from in vitro engineered Saccharomyces cerevisiae tRNA(Trp) genes.

Nonsense suppressors derived from Saccharomyces cerevisiae tRNA(Trp) genes have not been identified by classical genetic screens, although one can construct efficient amber (am) suppressors from them by making the appropriate anticodon mutation in vitro. Herein, a series of in vitro constructed putative suppressor genes was produced to test if pre-tRNA(Trp) processing difficulties could help to explain the lack of classical tRNA(Trp)-based suppressors. It is clear that inefficient processing of introns from precursor tRNA(Trp), or inaccurate overall processing, may explain why some of these constructs fail to promote nonsense suppression in vivo. However, deficient processing must be only one of the reasons why classical tRNA(Trp)-based suppressors have not been characterized, as suppression may still be extremely weak or absent in instances where the in vitro construct can lead to an accumulation of mature tRNA(Trp). Furthermore, suppression is also very weak in strains transformed with an intronless derivative of a putative tRNA(Trp) ochre (oc) suppressor gene, wherein intron removal cannot pose a problem.

Base Sequence↗

PubMed1989

Effect of variation of charged and uncharged tRNA(Trp) levels on ppGpp synthesis in Escherichia coli.

We introduced into a stringent Escherichia coli tryptophan auxotroph a plasmid bearing the tRNA(Trp) gene under the control of an inducible promoter. This allows us to manipulate the total concentration of tRNA(Trp) in the cell according to whether and when inducer is added to the culture. We also manipulated the concentration of Trp-tRNA(Trp) in vivo since the strain used bears a mutation in the Trp-tRNA synthetase affecting the Km for tryptophan, such that varying the exogenous concentration of tryptophan led to variation in the level of Trp-tRNA(Trp) in the cell. With this system, we found that the signal eliciting ppGpp synthesis during a stringent response triggered by tryptophan limitation did not depend on the absolute concentration of either charged or uncharged tRNA(Trp) but rather depended on a decline in the ratio of charged/uncharged tRNA(Trp). In addition, we found that the amplitude of the response, once triggered by tryptophan limitation, was determined by the total concentration of tRNA(Trp) present in the cell (which is mostly uncharged at that point in time). However, excess uncharged tRNA(Trp) did not amplify ppGpp synthesis triggered by limitation of a different amino acid. These data provide in vivo support for the in vitro-derived model of ppGpp synthesis on ribosomes.

Bacterial Proteins↗

PubMed2001

Species-specific differences in the operational RNA code for aminoacylation of tRNA(Trp).

Identity elements play essential roles in the recognition of tRNAs by their cognate aminoacyl-tRNA synthetase. An operational RNA code relates amino acids to specific sequences and structural features of tRNA acceptor stems. In this study, a series of tRNA(Trp) variants was prepared by in vitro transcription and their efficiencies of aminoacylation by tryptophan (k(cat)/K(m)) were measured with the aid of Bacillus subtilis and human tryptophanyl-tRNA synthetases (TrpRS). The identity elements in the operational RNA code of human tRNA(Trp) were found to be: major element, discriminator base A73; minor elements, G1/C72 and U5/G68. From the cross-species aminoacylation assays, we conclude that the identity elements in tRNA(Trp) from B.subtilis and human all contribute to species-specific aminoacylation by TrpRS. Analyses of 22 TrpRS sequences covering three taxonomic domains (bacteria, eukarya and archaea) reveal that the sequences are divided into two evolutionarily distant groups. The same partition is also observed in the analyses of tRNA(Trp) acceptor stem sequences. Our data suggest that the two TrpRS groups may reflect co-adaptations needed to accommodate changes in the operational RNA code for tryptophan.

Acylation↗

PubMed1992

The leaky UGA termination codon of tobacco rattle virus RNA is suppressed by tobacco chloroplast and cytoplasmic tRNAs(Trp) with CmCA anticodon.

RNA-1 molecules from tobacco rattle virus (TRV) and pea early-browning virus (PEBV), two members of the tobravirus group, have recently been shown to contain internal, in-frame UGA termination codons which are suppressed in vitro. Our results suggest that a UGA stop codon also exists in RNA-1 of pepper ringspot virus (PRV), another tobravirus. UGA suppression may therefore be a universal feature of the expression of tobravirus genomes. We have isolated two natural suppressor tRNAs from uninfected tobacco plants on the basis of their ability to promote readthrough over the leaky UGA codon of TRV RNA-1 in a wheat germ extract depleted of endogenous mRNAs and tRNAs. Their amino acid acceptance and nucleotide sequences identify the two UGA-suppressor tRNAs as chloroplast (chl) and cytoplasmic (cyt) tryptophan-specific tRNAs with the anticodon CmCA. These are the first UGA suppressor tRNAs to be identified in plants. They have several interesting features. (i) Chl tRNA(Trp) suppresses the UGA stop codon more efficiently than cyt tRNA(Trp). (ii) Chl tRNA(Trp) contains an A24:U11 pair in the D-stem as does the mutated Escherichia coli UGA-suppressor tRNA(Trp) which is a more active suppressor than wild-type tRNA(Trp). (iii) The suppressor activity of chl tRNA(Trp) is dependent on the nucleotides surrounding the stop codon because it recognizes UGA in the TRV context but not the UGA in the beta-globin context.

Animals↗

PubMed2006

Dual targeting of a single tRNA(Trp) requires two different tryptophanyl-tRNA synthetases in Trypanosoma brucei.

The mitochondrion of Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by the import of a small fraction of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single-copy genes, suggesting the use of the same enzyme in the cytosol and mitochondrion. However, the T. brucei genome contains two distinct genes for eukaryotic tryptophanyl-tRNA synthetase (TrpRS). RNA interference analysis established that both TrpRS1 and TrpRS2 are essential for growth and required for cytosolic and mitochondrial tryptophanyl-tRNA formation, respectively. Decoding the mitochondrial tryptophan codon UGA requires mitochondria-specific C-->U RNA editing in the anticodon of the imported tRNA(Trp). In vitro charging assays with recombinant TrpRS enzymes demonstrated that the edited anticodon and the mitochondria-specific thiolation of U33 in the imported tRNA(Trp) act as antideterminants for the cytosolic TrpRS1. The existence of two TrpRS enzymes, therefore, can be explained by the need for a mitochondrial synthetase with extended substrate specificity to achieve aminoacylation of the imported thiolated and edited tRNA(Trp). Thus, the notion that, in an organism, all nuclear-encoded tRNAs assigned to a given amino acid are charged by a single aminoacyl-tRNA synthetase, is not universally valid.

Amino Acid Sequence↗

PubMed1991

Copy number and chromosomal location of Saccharomyces cerevisiae tRNA(Trp)-encoding genes.

Yeast tRNA(Trp)-encoding genes have been identified by Southern-blot analysis of chromosomal DNA. Seven copies of this gene are detected in blots of both restriction endonuclease digests and intact chromosomal DNA. Electrophoretic mapping of tDNA(Trp) indicates one copy is located on each of chromosomes X, XI, XIII, and XVI. The remaining three copies are localized to chromosomes VII and/or XV. Three different yeast strains gave identical results indicating this multi-gene family is relatively stable.

Base Sequence↗

PubMed1998

tRNATrp as a key element of antitermination in the Lactococcus lactis trp operon.

The expression of the trp operon of Lactococcus lactis is regulated in response to tryptophan availability by a mechanism of transcription antitermination. We present evidence in support of a previously described model involving tRNATrp as a key element in the sensing of tryptophan levels and the realization of the regulatory response to tryptophan limitation. In agreement with this model, two sites of presumed direct interaction between the trp leader transcript and tRNATrp are found to be of crucial importance for efficient antitermination. These correspond to the specifier codon, which presumably interacts with the anticodon in the tRNA, and a sequence complementary to, and presumably interacting with, the acceptor stem of the tRNA. Through these interactions, uncharged tRNATrp is believed to stabilize an antiterminator conformation of the trp leader transcript, thus allowing transcription and expression of the structural genes of the operon. For the first time, we present direct evidence that it is the ratio of uncharged to charged tRNA that is important for the regulation of antitermination, rather than the absolute amount of uncharged tRNA. In addition, our results indicate that the codon-anticodon interaction, although contributing largely to the efficiency of the regulatory response, is not strictly indispensable, which suggests the existence of additional interactions between mRNA and tRNA. Finally, we describe a possible additional level of regulation, superimposed and dependent on tRNA-mediated anti-termination control, that is based on the processing of the trp leader transcript. Together with the regulation mechanisms described earlier for the Escherichia coli and Bacillus subtilis trp operons, this constitutes the third different mechanism of transcript elongation control found to be involved in the regulation of an operon of which the structural genes are highly conserved.

Artificial Gene Fusion↗

PubMed1998

Effect of B. subtilis TRNA(Trp) on readthrough rate at an opal UGA codon.

Bacillus subtilis has been thought to have a high readthrough rate at the UGA stop codon because no opal suppressor tRNA has been isolated so far [Lovett et al. (1991) J. Bacteriol. 173, 1810-1812]. To examine whether a tRNATrp which we have characterized [Matsugi et al. (1992) Nucleic Acids Res. 20, 3514] has the ability to read the UGA codon, in vitro translation was performed with a synthetic mRNA containing a test codon, UGA, UAG, UAA, or UGG, in a reading frame. Addition of Trp-tRNATrp to the system significantly increased the readthrough rate only in the case of UGA. This suggests that this tRNATrp has a dual recognition pattern in B. subtilis, i.e., for the canonical tryptophan codon and for readthrough at the UGA stop codon.

Anticodon↗

PubMed2004

A tRNA(TRP) gene mediates the suppression of cbs2-223 previously attributed to ABC1/COQ8.

The Saccharomyces cerevisiae gene ABC1 was originally isolated as a multicopy suppressor of a yeast strain harboring a mutation in a cytochrome b translational activator (cbs2-223). Based on this identification, Abc1p was postulated to activate the bc1 complex and function as a chaperone of cytochrome b. ABC1 was subsequently identified as COQ8 and found to be necessary for yeast coenzyme Q synthesis. In this work we show that a segment of yeast genomic DNA containing ABC1/COQ8 and neighboring genes suppresses the respiratory and Q-deficient phenotypes of the coq6 mutant, coq6-1. COQ6 is essential for yeast coenzyme Q biosynthesis. We show that a tRNA(TRP) gene located downstream of ABC1/COQ8 mediates suppression of the cbs2-223 and coq6-1 mutations, and each is identified here as containing UGA nonsense codons. The inability of ABC1/COQ8 to suppress the cbs2-223 allele in multicopy indicates it may not be a chaperone as previously reported.

Gene Expression Regulation, Fungal↗