Families Of tRNAs Research Articles

Mistranslation of the genetic code by a new family of bacterial transfer RNAs

The correct coupling of amino acids with transfer RNAs (tRNAs) is vital for translating genetic information into functional proteins. Errors during this process lead to mistranslation, where a codon is translated using the wrong amino acid. While unregulated and prolonged mistranslation is often toxic, growing evidence suggests that organisms, from bacteria to humans, can induce and use mistranslation as a mechanism to overcome unfavorable environmental conditions. Most known cases of mistranslation are caused by translation factors with poor substrate specificity or when substrate discrimination is sensitive to molecular changes such as mutations or posttranslational modifications. Here we report two novel families of tRNAs, encoded by bacteria from the Streptomyces and Kitasatospora genera, that adopted dual identities by integrating the anticodons AUU (for Asn) or AGU (for Thr) into the structure of a distinct proline tRNA. These tRNAs are typically encoded next to a full-length or truncated version of a distinct isoform of bacterial-type prolyl-tRNA synthetase. Using two protein reporters, we showed that these tRNAs translate asparagine and threonine codons with proline. Moreover, when expressed in Escherichia coli, the tRNAs cause varying growth defects due to global Asn-to-Pro and Thr-to-Pro mutations. Yet, proteome-wide substitutions of Asn with Pro induced by tRNA expression increased cell tolerance to the antibiotic carbenicillin, indicating that Pro mistranslation can be beneficial under certain conditions. Collectively, our results significantly expand the catalog of organisms known to possess dedicated mistranslation machinery and support the concept that mistranslation is a mechanism for cellular resiliency against environmental stress.

Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu

Transcription and translation of mRNA's are coordinated processes in bacteria. We have previously shown that a mutant form of EF-Tu (Gln125Arg) in Salmonella Typhimurium with a reduced affinity for aa-tRNA, causes ribosome pausing, resulting in an increased rate of RNase E-mediated mRNA cleavage, causing extremely slow growth, even on rich medium. The slow growth phenotype is reversed by mutations that reduce RNase E activity. Here we asked whether the slow growth phenotype could be reversed by overexpression of a wild-type gene. We identified spoT (encoding ppGpp synthetase/hydrolase) as a gene that partially reversed the slow growth rate when overexpressed. We found that the slow-growing mutant had an abnormally high basal level of ppGpp that was reduced when spoT was overexpressed. Inactivating relA (encoding the ribosome-associated ppGpp synthetase) also reduced ppGpp levels and significantly increased growth rate. Because RelA responds specifically to deacylated tRNA in the ribosomal A-site this suggested that the tuf mutant had an increased level of deacylated tRNA relative to the wild-type. To test this hypothesis we measured the relative acylation levels of 4 families of tRNAs and found that proline isoacceptors were acylated at a lower level in the mutant strain relative to the wild-type. In addition, the level of the proS tRNA synthetase mRNA was significantly lower in the mutant strain. We suggest that an increased level of deacylated tRNA in the mutant strain stimulates RelA-mediated ppGpp production, causing changes in transcription pattern that are inappropriate for rich media conditions, and contributing to slow growth rate. Reducing ppGpp levels, by altering the activity of either SpoT or RelA, removes one cause of the slow growth and reveals the interconnectedness of intracellular regulatory mechanisms.

BayesFold: Rational 2° folds that combine thermodynamic, covariation, and chemical data for aligned RNA sequences

BayesFold is a Web application that folds an alignment of closely related sequences and evaluates hypotheses about their shared structure. It uses Bayes's Theorem to combine information from several sources, including chemical mapping (if available), thermodynamic folding, and observed sequence variations. Its method provides a rational basis for integrating results, even when these methods conflict. On a gapped alignment of 86 tRNAPhe sequences each 77 bases long, BayesFold takes 31 sec to perform the calculations; the best structure contained 95% of the base pairs in the true structure, and the true structure was ranked second. Notably, similar results come from random samples of only 10 sequences from the alignment (running time 3 sec), suggesting that remarkably few sequences are required for good results. In contrast, folding single sequences with BayesFold produced structures 9.6 bp different, or with the Vienna package, 13.4 bp different, from the true structure. Similar results were obtained for other families of tRNAs. We especially recommend BayesFold for alignments of 3-50 closely related sequences, such as the sequence families frequently found in SELEX. In addition to providing a convenient way to explore the effects of each of the criteria on the plausibility of different structures, BayesFold also makes it easy to produce publication-quality secondary-structure graphics. The Web interface, available at http://bayes.colorado.edu/fold/, includes the flexibility to thread any of the sequences (or the consensus sequence) through any of the structures, including the one judged most probable.

SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

Why is tumor tRNA hypomodified with respect to Q nucleoside?

Nucleoside Q is found exclusively in the first position of the anticodon in tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp (Kasai et al. 1975b, 1976). Its function is unknown, and long-term Q-deficient, germ-free mice appear normal (Reyniers et al. 1981). However, queuirie metabolism is altered in neoplasia. Many tumors exhibit Q-deficient tRNA (Katze 1975; Okada et al. 1978; Roe et al. 1979) and queuine addition to tumor-bearing mice can reverse tumor tRNA Q deficiency (Katze and Beck 1980; Nishimura et al., this volume pp. 401–412) and may inhibit tumor growth (Katze and Beck 1980). It is unlikely that Q-deficient tRNA is necessary for the neoplastic state since the careful study of Marini and Mushinski (1979; Mushinski and Marini, this volume pp. 121–132) shows widely varying, repeatable isoaccepting patterns (largely due to differences in Q content) for the Q family of tRNAs from several Balb/c mouse plasmacytomas with similar growth rates and size at harvest. These large differences among such a similar group of tumors suggest that the alteration in Q metabolism is secondary to the neoplastic process.

Threonine tRNAs and their genes in Escherichia coli

The subject of this study was the threonine isoacceptor family of tRNAs in Escherichia coli and the genes coding for them. The goal was to identify and map all the genes and to determine the relative contribution of each gene to the tRNA pool. The mapping experiments exploited gene-dosage effects in partially diploid strains; if a strain harboring a particular F' episome overproduced a particular tRNA species, it could be concluded that the gene for that tRNA was located on the chromosomal segment carried by the F'. Isoacceptor tRNAs were distinguished by column fractionation. It was found that there are three major threonine tRNA species that occur in roughly equal amounts. These are tRNAThr1, which is encoded by a gene in the distal region of the rrnD ribosomal RNA operon, and tRNAThr3 and tRNAThr4, which comes from genes in the cluster thrU tyrU glyT thrT at 89 min on the map. The relative abundances of the tRNA species roughly match the reported frequencies of the codons that they recognize in mRNA. Although the tRNAThr4 has a mismatched base pair that raised questions about its biological activity, it was found to be functional at least with respect to recognition by the threonyl-tRNA synthetase. An apparent fourth gene affecting threonine tRNA has been identified and mapped at 6-8 min; it is here designated thrW. It may be a structural gene for a minor tRNA species, present in one-third the amount of each of the major species, and chromatographically indistinguishable from tRNAThr4.

Effect of diet on the queuosine family of tRNAs of germ-free mice.

The transfer RNAs for aspartic acid, asparagine, histidine, and tyrosine respond to codons in the third column of the genetic code and contain a hypermodified nucleoside known as queuosine (Q) in the first position of the anticodon of the major isoacceptor tRNA. Nothing is known about the physiological or biochemical function of Q. Germ-free mice were maintained for a period of nine tRNA half-lives on a chemically defined diet known to contain all essential constituents of the rodent diet but no Q or its base, queuine. The tRNAs for histidine and asparagine contained only 15% of the Q-containing isoacceptor tRNA. On the other hand, the Q-containing isoacceptor comprised 88% of the tRNAHis and 85% of the tRNAAsn in conventional mice and germ-free mice fed commercial mouse chow. Transfer RNAAsp and tRNATyr were completely modified with respect to Q in germ-free mice maintained on the chemically defined diet as well as on normal mouse chow. Germ-free mice fed the chemically defined diet contained normal amounts of the hypermodified base wye in tRNAPhe.

An analysis of Q and Q ∗ containing tRNAs during the development of Lucilia sericata, Musca domestica and Tenebrio molitor

The chromatographic profiles of the family of tRNAs containing the hypermodified nucleoside Q and Q ∗ in the first position of their anticodons were examined in the third instar larvae and adults of Lucilia sericata and Musca domestica. The absolute amounts of these tRNAs remained constant during development relative to phenylalanine tRNA. In M. domestica only Q containing forms were found in both larvae and adults while in L. sericata, the larvae contained a significant portion of the Q lacking form. In Tenebrio molitor the Q lacking form of tyrosine tRNA appeared to be only significant in pupae. The developmental patterns of Q biosynthesis in the Diptera therefore range from almost entirely Q containing in M. domestica to entirely Q lacking in Drosophila raised under certain conditions. This suggests that this modification to the anticodon is not playing a common regulatory function in the development of these insects.

Incorrect aminoacylations involving tRNAs or valyl-tRNA synthetase from Bacillus stearothermophilus.

Numerous misacylations occur on heterologous systems containing unfractionated tRNAs from yeast or from Bacillus stearothermophilus and pure valyl-tRNA synthetase from B. stearothermophilus or from yeast, when special aminoacylation conditions are used. In the homologous system and in heterologous systems where the unfractionated tRNAs and the enzyme originate from prokaryotic organisms (B. stearothermophilus and Escherichia coli), the errors are seldom. This phenomenon is explained by competition effects between the cognate tRNAVal and non-cognate tRNAs for the valyl-tRNA synthetase. But using pure tRNA species, errors can be observed in such systems, even under classical assay conditions; in particular it was shown that the valyl-tRNA synthetase from B. stearothermophilus catalyses the misacylation of E. coli tRNAIle and tRNAtMet and of yeast tRNAtMet and tRNAPhe. These reactions are characterized by Km values slightly increased as compared to the value obtained in the cognate system and by V values decreased by a factor of about 40 to 3000 compared to the cognate tRNA species. In the presence of dimethylsulfoxide, the rate and the extent of those misacylation reactions are enhanced. In the case of E. coli tRNAPhe, the misacylation occurs only in the presence of the organic solvent. In no case however, new aminoacylation errors are induced at high temperature (50–75 °C) in the presence of the thermostable valyl-tRNA synthetase from B. stearothermophilus; only an increase of the rates of the aminoacylation reactions which already occur at 30 °C has been observed at higher temperature. Thus organic solvent and heat must have distinct effects on the essential parameters determining the specificity of the tRNA aminoacylation reactions. Also it has been observed that the most easily misacylated tRNA species by valyl-tRNA synthetase from B. stearothermophilus are the same as those which are misacylated by the valyl-tRNA synthetases from E. coli and from yeast. This observation suggests the existence of a family of tRNAs containing besides of tRNAVal, other tRNAs such as tRNAIle, tRNAtMet and tRNAPhe, and which are likely to be related from a phylogenic point of view. Moreover these tRNA species have also been found to be easily misacylated by other aminoacyl-tRNA synthetases namely the enzymes specific for isoleucine and phenylalanine, thus suggesting more generally the existence of interacting tRNA-aminoacyl-tRNA synthetase families.

The Variety of Intraspecific Misacylations Carried Out by Isoleucyl Transfer Ribonucleic Acid Synthetase of Escherichia coli

Abstract The variety of tRNAs (Escherichia coli) misisoleucylated by isoleucyl-tRNA synthetase in normal and mixed solvents is examined. The method employs microscale amino acid radioanalysis, used to detect release of cognate amino acids from tRNAs which have been previously protected from periodate by misisoleucylation. In the presence of unresolved acceptor tRNAs highly purified isoleucyl-tRNA synthetase simultaneously misacylates several E. coli B species in addition to those previously studied (tRNAPhe (Yarus, M. (1972) Biochemistry 11, 2352) and tRNAMet (Mertes, M., Peters, M.A., Mahoney, W., and Yarus, M. (1972) J. Mol. Biol. 71, 671)). There is a tendency for tRNAs cognate to chemically similar amino acids to be more easily misacylated. Some reactions are detectable in both normal and mixed solvents (20% methanol). In contrast to tRNAPhe and tRNAMet, the misacylation of some tRNAs is not responsive to addition of methanol. Thus, some intercognate barriers are lowered in mixed solvents, and some are not. This suggests that isoleucyl-tRNA synthetase distinguishes tRNAs by at least two molecular mechanisms. Comparison of these data with the experiments of other laboratories enables one to descry a family of tRNAs, defined as that set of acceptor species most easily misacylated by several aminoacyl-tRNA synthetases cognate to hydrophobic amino acids.

Purification of Five Serine Transfer Ribonucleic Acid Species from Escherichia coli and Their Acylation by Homologous and Heterologous Seryl Transfer Ribonucleic Acid Synthetases

can esterify serine to all of the purified isoacceptor RNAs. However, the extent of charging is only a small fraction of the level obtained with the homologous seryl-tRNA synthetase. It has been well established that most amino acids are speci- fied by more than one codon. A single tRNA molecule, how- ever, can recognize only one, two, or three codons. Thus, the proper translation of the genetic message to an amino acid specified by four codons, for example, requires at least two tRNA species (1, 2). This explains the occurrence in cells of families of tRNAs with different coding response but specifying the same amino acid (isoaccepting tRNAs). The number of such isoacceptors is further increased by the existence of multiple tRNA species specific for the same ammo acid and the same codon (redundant tRNAs (2)). Since leucine, serine, and arginine each have six codons, a larger number of isoacceptor RNAs is expected for these amino acids than for all others. Of these, the family of serine tRNAs has been most carefully studied, and there are three reports (3-5) of separation and partial puri- fication of the tRNASer family from Escherichia coli. This report describes a more extensive separation and purifi- * This work was supported by Grants GM 15401 and FR-07015 from the United States Public Health Service, National Institutes of Health, and Grant GB 7269 from the National Science Foun- dation. $ Centennial Fellow