Isoacceptor Species Research Articles

Bacterial tRNA 2'-O-methylation is dynamically regulated under stress conditions and modulates innate immune response.

RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2′-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS show substantial, but not uniform, increase of the Gm18 level in selected tRNAs under mild bacteriostatic antibiotic stress, while other Nm modifications remain relatively constant. The absence of Gm18 modification in tRNAs leads to moderate alterations in E. coli mRNA transcriptome, but does not affect polysomal association of mRNAs. Interestingly, the subset of motility/chemiotaxis genes is significantly overexpressed in ΔTrmH mutant, this corroborates with increased swarming motility of the mutant strain. The stress-induced increase of tRNA Gm18 level, in turn, reduced immunostimulation properties of bacterial tRNAs, which is concordant with the previous observation that Gm18 is a suppressor of Toll-like receptor 7 (TLR7)-mediated interferon release. This documents an effect of stress induced modulation of tRNA modification that acts outside protein translation.

Modulation of differential transcription of tRNA genes through chromatin organization

In higher eukaryotes, tRNA multigene families comprise several copies encoding the same tRNA isoacceptor species. Of the 11 copies of a tRNA1Gly family from the mulberry silkworm Bombyx mori, individual members are differentially transcribed in vivo in the B. mori-derived BmN cell lines and in vitro in silk gland nuclear extracts. These genes have identical coding regions and hence harbour identical internal control sequences (the A and B boxes), but differ significantly in their 5' and 3' flanking regions. In the present study, we demonstrate the role of chromatin structure in the down-regulation of the poorly expressed copy, tRNA1Gly-6,7. Distinct footprints in the 5'-upstream region of the poorly transcribed gene in vitro as well as in vivo suggested the presence of nucleosomes. A theoretical analysis of the immediate upstream sequence of this gene copy also revealed a high propensity of nucleosome formation. The low transcription of tRNA1Gly-6,7 DNA was further impaired on assembly into chromatin and this inhibition was relieved by externally supplemented TFIIIC with an associated histone acetyltransferase activity. The inhibition due to nucleosome assembly was absent when the 5'-upstream region beyond -53 nt was deleted or entirely swapped with the 5'-upstream region of the highly transcribed gene copy, which does not position a nucleosome. Footprinting of the in vitro assembled tRNA1Gly-6,7 chromatin confirmed the presence of a nucleosome in the immediate upstream region potentially masking TFIIIB binding. Addition of TFIIIC unmasked the footprints present on account of the nucleosome. Our studies provide the first evidence for nucleosomal repression leading to differential expression of individual members from within a tRNA multigene family.

Enhanced expression of initiator TRNA(Met) in human gastric and colorectal carcinoma.

Transfer RNA isoaccepting species are differentially expressed at different times during development, differentiation, growth, aging, and carcinogenesis processes. It has been suggested that alterations in tRNA patterns might be mechanistically important in modulating gene expression during the various physiological/pathological cellular stages. As part of a study to investigate the possible mechanisms by which alterations of translational machinery can start and/or sustain carcinogenic cell proliferation, in this communication we report analysis of tRNA distribution in two gastro-intestinal human tumors. The qualitative and quantitative data obtained for cellular tRNA distribution put into evidence a shift in the tRNA population with increased level of initiator tRNA(Met) in the malignant tissues. This observation confirms previous data obtained on experimental carcinogenesis models and suggests the possibility of specific involvement of tRNA changes in protein synthesis initiation during tumorigenesis.

Separation of Rat Liver Cytoplasmic Trnas by High Performance Liquid Chromatography

Abstract Experiments aimed at developing a chromatographic map of cytoplasmic tRNA population from rat liver are described. HPLC tRNA fractionation by a linear gradient of acetonitrile/ammonium acetate, results in a chromatogram with more than 60 significant peaks, having area peak percentages ranging from 0.001 to 5.0. This paper presents evidence that each peak corresponds to a specific tRNA species. Specificity of tRNA-peak assignment was established by charging total tRNA with a mixture of 19 unlabeled aminoacids plus a radioactive aminoacid, and isolating the radioactive aminoacylated iso-tRNAs by gel electrophoresis, followed by HPLC acetonitrile gradient. The radioactive aminoacyl-tRNA species were eluted as well-behaved peaks. Here we describe the analysis of tRNATrp, which gave 2 well separated radioactive peaks, most likely corresponding to tRNATrp isoacceptor species postulated by the wobble hypothesis. Taken together, the present data validate this procedure for monitoring quantitatively and q...

Fractionation of rat liver tRNA by reversed-phase high performance liquid chromatography: isolation of Iso-tRNAs(Pro).

This paper illustrates the fractionation of cytoplasmic transfer ribonucleic acid from rat liver by reversed-phase high performance liquid chromatography using a gradient of acetonitrile/ammonium acetate. The procedure is fast, highly reproducible, and gives an excellent resolution of the numerous tRNA population: about 50 peaks with area peak percentages ranging from 0.001 to 5 can be monitored. Uncharged tRNA preparations exhibited a chromatographic profile different from aminoacylated tRNA, thus suggesting a possible strategy to distinguish between aminoacylated and nonacylated tRNA species. Moreover, a first approach to map the HPLC peaks was attempted by chromatographing preparations of tRNA which had been aminoacylated with individual 3H-labeled aminoacids. Here is reported the case of tRNA(Pro), which gave three well separated radioactive peaks, most likely corresponding to tRNA(Pro) isoacceptor species.

Growth-rate-dependent Accumulation of Twelve tRNA Species in Escherichia coli

We have previously shown that in Escherichia coli the accumulation of five leucine and three methionine tRNA species is regulated so that those tRNA species that translate major codons increase while those that translate minor codons decrease as the growth rate increases. Here, we have analyzed the growth-rate-dependence of another 12 tRNA species. We find that the level of three tRNA species cognate to the major glycine, proline and arginine codons, respectively, increase with increasing growth rates. Conversely, four tRNAs that are cognate to minor codons within the same amino acid families decrease with increasing growth rates. In addition, the glutamyl as well as the phenylalanyl isoacceptor species are accumulated in proportion to the content of these two amino acids in the proteins produced at different growth rates. In summary, the patterns of the growth-rate-dependence for the accumulation of these 17 tRNA species support the interpretation that the major codon preference is an arrangement to maximize the growth rates of bacteria in rich media by optimizing the kinetic efficiency of translation. In contrast, we find that three minor tRNA species cognate to two rare arginine codons and one minor glycine codon, respectively, increase with increasing growth rate. Such findings suggest that there are additional constraints on the accumulation of these tRNA species that may be distinct from those required to optimize the kinetic efficiency of translation.

Editing does not exist for mammalian selenocysteine tRNAs.

It has been reported that selenocysteine tRNA from bovine liver is completely edited to two isoacceptor species, called tRNA([Ser]SecNCA) and tRNA([Ser]SecCmCa), which differ from the gene sequence. We used direct tRNA sequencing, mobility shift analyses, primer extension, restriction enzyme digestion and single strand conformational polymorphism (SSCP) analyses of products from reverse transcription coupled with polymerase chain reaction (RT/PCR), sequencing of RT/PCR products and HPLC-coupled mass spectrometry to reproduce this result and show here that editing of these tRNAs does not occur.

The first human genes for tRNA(ArgICG), tRNA(GlyUCC), and tRNA(ThrIGU) and more tRNA(Val) pseudogenes: expression and pre-tRNA maturation in HeLa cell-free extracts.

A functional tRNA(Val) gene, which codes for the major tRNA(ValIAC) isoacceptor species, and three new tRNA(Val) pseudogenes have been isolated from human genomic DNA. Two tRNA(Val) pseudogenes and a tRNA(Val) variant gene were found to be associated with tRNA genes encoding tRNA(ArgICG), tRNA(GlyUCC), and tRNA(ThrIGU), respectively, on distinct DNA fragments. All tRNA genes, including the pseudogenes, are actively transcribed in HeLa nuclear extract. Pre-tRNAs of tRNA(Val), tRNA(Arg), tRNA(Thr), and tRNA(Gly) genes are correctly processed to mature-sized tRNAs, whereas the three tRNA(Val) pseudogenes yield stable pre-tRNAs in vitro. These findings reveal that, together with the three known pseudogenes, half of the members of the human tRNA(Val) gene family are pseudogenes, all of which are active in homologous nuclear extracts in vitro and presumably also in vivo.

An extreme codon preference strategy: codon reassignment.

We argue that in animal mitochondria codon reassignments, such as those for AGA and AGG from arginine to serine or of AUA from isoleucine to methionine, are the result of an interplay between biased mutational forces and selective ones. In particular, there is a marked tendency for animal mitochondria to have very small genomes and to minimize their investment in components required for gene expression. These tendencies are expressed as a reduction in the diversity of tRNA isoacceptor species. In our view, the pressure to simplify tRNA populations, together with mutational bias against certain codons, will account for the codon reassignments observed in animal mitochondria. A parallel to the major codon bias in microorganisms, which likewise tends to reduce the diversity of the tRNA isoacceptor populations under fast growth conditions, may be drawn. Therefore, we suggest that codon reassignments are usefully viewed as an extreme form of codon bias.

Genes, variant genes, and pseudogenes of the human tRNAValgene family are differentially expressed in HeLa cells and in human placenta

Pre-tRNAs(Val) were identified in unfractionated tRNA preparations from HeLa cells and human placenta and their 5' leader structures were deduced from the nucleotide sequences of the corresponding cDNAs. Several of these precursors can be assigned to nine out of the eleven members of the human tRNA(Val) gene family characterized so far, which demonstrates that these gene loci are actively transcribed in vivo. Among the expressed genes there are (a) genes for the two known tRNA(Val) isoacceptor species from human placenta, (b) gene variants that exhibit sequence alterations as compared to conventional genes, and (c) pseudogenes that produce processing-deficient precursors which are not matured to tRNAs. The transcription products of several yet unknown tRNA(Val) genes have also been detected. Furthermore, different expression patterns are observed in the two cell types studied. These data allow for the first time an insight into the in vivo expression of a human tRNA gene family.

TRNA-like structures and gene regulation at the translational level: a case of molecular mimicry in Escherichia coli.

Escherichia coli threonyl-tRNA synthetase regulates the translation of its own mRNA by binding to it in a region, called the operator, located in front of the ribosomal binding site. The primary and secondary structures of the operator resemble those of the anticodon arm of several tRNA(Thr) isoacceptor species. We reasoned that if the interaction between the synthetase and its two partially analogous ligands, the tRNA and the mRNA, had some common features, single mutations in the enzyme should affect both interactions in a very similar way. We thus isolated synthetase mutants (called super-repressors) that repress the translation of their mRNA in trans to an extreme level, and other mutants that are completely unable to perform any repression. The super-repressors, which are suspected to bind their mRNA with high affinity, are shown to bind the tRNA with an increased affinity. The non-repressing mutants, which are suspected to have lost their capacity to bind the mRNA, are shown to bind their tRNA with less affinity. The binding properties of the mutant enzymes for the other substrates, ATP and threonine, are unchanged. The observed correlation between regulatory and aminoacylation defects strongly suggests that the synthetase recognizes the similar parts of its two RNA ligands--the anticodon-like arm of the mRNA and the true anticodon arm of the tRNA--in an analogous way.

Missense and nonsense suppressors can correct frameshift mutations

Missense and nonsense suppressor tRNAs, selected for their ability to read a new triplet codon, were observed to suppress one or more frameshift mutations in trpA of Escherichia coli. Two of the suppressible frameshift mutants, trpA8 and trpA46 AspPR3, were cloned, sequenced, and found to be of the +1 type, resulting from the insertion of four nucleotides and one nucleotide, respectively. Twenty-two suppressor tRNAs were examined, 20 derived from one of the 3 glycine isoacceptor species, one from lysT, and one from trpT. The sequences of all but four of the mutant tRNAs are known, and two of those four were converted to suppressor tRNAs that were subsequently sequenced. Consideration of the coding specificities and anticodon sequences of the suppressor tRNAs does not suggest a unitary mechanism of frameshift suppression. Rather, the results indicate that different suppressors may shift frame according to different mechanisms. Examination of the suppression windows of the suppressible frameshift mutations indicates that some of the suppressors may work at cognate codons, either in the 0 frame or in the +1 frame, and others may act at noncognate codons (in either frame) by some as-yet-unspecified mechanism. Whatever the mechanisms, it is clear that some +1 frameshifting can occur at non-monotonous sequences. A striking example of a frame shifting missense suppressor is a mutant lysine tRNA that differs from wild-type lysine tRNA by only a single base in the amino acid acceptor stem, a C to U70 transition that results in a G·U base pair. It is suggested that when this mutant lysine tRNA reads its cognate codon, AAA, the presence of the G·U base pair sometimes leads either to a conformational change in the tRNA or to an altered interaction with some component of the translation machinery involved in translocation, resulting in a shift of reading frame. In general, the results indicate that translocation is not simply a function of anticodon loop size, that different frameshifting mechanisms may operate with different tRNAs, and that conformational features, some far removed from the anticodon region, are involved in maintaining fidelity in translocation.

Chromatography ofE. colitRNA on 5–20 μm Agarose Beads

Abstract Escherichia coli transfer RNAs have been separated by chromatography on 5–20 μm beads of divinylsulfone-crosslinked agarose with the use of isocratic elution combined with a negative salt gradient. For example, tRNALeu was resolved into six isoacceptor species and tRNASer into four. Leucine-charged tRNA's were eluted after their corresponding uncharged isoacceptor species. By optimizing flow rate, column length and elution buffer the fractionation of tRNA could be performed within 3 hours.

Specific occurrence of selenium in enzymes and amino acid tRNAs 1

In contrast to the widespread ability of bacteria, plants, and animals to incorporate selenium nonspecifically into proteins in the form of selenomethionine residues, the selenoamino acid selenocysteine occurs as a highly specific component of a few selenium-dependent enzymes. Selenocysteine has been identified in glycine reductase, formate dehydrogenase, and hydrogenase of bacterial origin and glutathione peroxidase from mammalian and avian sources. In these enzymes there is evidence that the selenol group, which is largely ionized at physiological pH, functions as a redox center. It now seems clear, from studies with both prokaryotes and eukaryotes, that the UGA opal stop codon is used to specify the cotranslational insertion of selenocysteine into proteins. The factors that allow this unusual use of the stop codon are, however, unknown. The occurrence of selenium as a normal constituent of several bacterial tRNA species has been established. The presence of a selenonucleoside, 5-methylaminomethyl-2-selenouridine, in the first or wobble position of the anticodons of certain glutamate and lysine iso-acceptor species influences codon-anticodon interaction and thus may serve to regulate translational processes. The biosynthesis of the selenonucleoside appears to involve the ATP-dependent activation of the sulfur in a preformed 5-methylaminomethyl-2-thiouridine residue in tRNA and replacement of the sulfur with selenium.

Structure of the Long Terminal Repeat of Simian Lymphotropic Virus Type III (African Green Monkey) and Its Relatedness to That of HIV

The simian T-lymphotropic virus type III (STLV-III[AGM]) is a retrovirus in wild African green monkeys which is serologically related to the human T-lymphotropic virus type III (HTLV-III/LAV-1/HIV) and other related human retroviruses. The long terminal repeats (LTR) contained in clones of viral DNA of (STLV-III[AGM]) were subcloned in M13 and their DNA sequence was determined and compared with that of HIV (HTLV-III[BH10]). The STLV-III(AGM) LTR is considerably larger than that of HTLV-III(BH10) (800 bp vs 634 bp) and contains a 498 bp U3 region, a 176 bp R region, and a 126 bp U5 region. These two LTR sequences share regions of significant homology. Regions of greatest homology include the 5' portion of U3, a core enhancer sequence in U3, sequences including and surrounding the TATAA promoter box in U3 and the AATAAA polyadenylation/termination signal in R, and the 3'-most region of U5. The relatively larger size of the STLV-III LTR is due to the presence in all three parts of the LTR of sequences which have no apparent homolog in the HIV LTR. Overall, the two LTRs are 47% homologous. Even greater homology (75%) is evident with a 300 bp segment including R and some of U3 from the LTR of another human retrovirus, HIV-2/LAV-2. The STLV-III LTR contains an imperfect 28 bp direct repeat in the R region which is not present in HIV. There are no obvious direct repeats in U3 homologous to the 10 bp repeat in the U3 of HTLV-III.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in the tRNA population between summer and winter acclimatized carp

1. 1. Transfer RNA isolated from the livers of winter and summer acclimatized male carps was fractionated by two-dimensional polyacrylamide electrophoresis. 2. 2. Significant changes in the distribution of the tRNA isoacceptor species were observed. 3. 3. The adjustment of the tRNA population to the needs of the protein synthetic machinery during the acclimatization process correlates with the cyclic carp liver cell ultrastructural changes previously observed, and provides a new approach to gain knowledge about the mechanisms involved in the compensatory response to thermal adaptation.

Plant tRNA suppressors: In vivo readthrough properties and nucleotide sequence of yellow lupin seeds tRNA Tyr

Transfer RNAs isolated from yellow lupin seeds were screened for tRNA dependent nonsense suppression in Xenopus laevis oocyte system. No opal (UGA) suppressor activity could be detected. However, a tRNA Tyr isoacceptor species stimulates readthrough of the leaky UAG tobacco mosaic virus (TMV)-RNA stop codon. This natural suppressor tRNA was purified to homogeneity and its sequence was determined to be: pCCg ACCUUAm 2GcUCAGDGGm GUAGAGCm 2 2GGAGGACUG ψAm 1GAψCCUUAGm 7Gacp 3UC ACUGGUψCGm 1AAUz.sbnd;CCG GUAGGUCGGACCA OH. The GψA anticodon seqeunce, the presence of the modified nucleoside 3-(3-amino-3-carboxypropyl)uridine (acp 3U) in the extra loo p and the absence of ribothymidine are of special interest.

Relative affinities of all Escherichia coli aminoacyl-tRNAs for elongation factor Tu-GTP.

The relative affinities of all Escherichia coli amino-acyl-tRNAs for E. coli elongation factor (EF) Tu-GTP have been measured by two independent applications of the competition form of the ribonuclease resistance assay. The set of aminoacyl-tRNAs includes at least one tRNA for each of the 20 amino acids as well as purified isoacceptor tRNA species for arginine, glycine, leucine, lysine, and tyrosine. In the first competition study, [3H]Phe-tRNA was used as the competing aminoacyl-tRNA against [14C]aminoacyl-tRNA in the set of all tRNAs; in the second study, [3H]Leu-tRNALeu4 was used as the competing aminoacyl-tRNA. The relative order of aminoacyl-tRNA affinities for EF-Tu-GTP was the same in each study. The results indicate that the affinity of EF-Tu-GTP at 4 degrees C, pH 7.4, is strongest for Gln-tRNA and weakest for Val-tRNA. Both Gly-tRNA and Pro-tRNA bind very strongly to EF-Tu-GTP relative to other aminoacyl-tRNAs. Various models of ternary complex interactions are discussed in light of the new data. Although the properties of the amino acid substituent are primarily responsible for the differences in relative affinities among the noninitiator aminoacyl-tRNAs, the results for the four isoacceptor species of Leu-tRNALeu indicate that the secondary structural features of the tRNA are also influential.

New biologic functions — Selenium-dependent nucleic acids and proteins

Selenium occurs normally in living things as a highly specific component of certain enzymes and amino acid transfer nucleic acids (tRNAs). In bacteria, biosynthesis of essential selenoenzymes has been shown to be unaffected by wide variations in sulfur levels. The naturally occurrring selenoenzymes so far identified from bacterial sources include glycine reductase, certain formate dehydrogenases, a hydrogenase, nicotinic acid hydroxylase, xanthine dehydrogenase and thiolase. The selenoenzyme, glutathione peroxidase, and three other selenoproteins of unknown function have been isolated from animals. In certain enzymes, e.g. glycine reductase, formate dehydrogenase, hydrogenase and glutathione peroxidase, the chemical form of selenium has been identified as selenocysteine. One enzyme, a bacterial thiolase, contains selenomethionine rather than selenocysteine. A labile, unidentified form of selenium is present in nicotinic acid hydroxylase, and by inference, xanthine dehydrogenase. The seleno-tRNAs serve as examples of a different type of biological macromolecule that is specifically modified with selenium. The major seleno-tRNAs in Clostridium sticklandii and Escherichia coli have been identified as glutamate and lysine isoaccepting species. The selenium-modified nucleoside is 5-methyl-aminomethyl-2-selenouridine (mnm 5Se 2U), which is the chemical analog of 5-methylaminomethyl-2-thiouridine, a previously identified minor base of E. coli tRNA 2 Glu. The seleno-tRNA Glu of C. sticklandii contains one gram atom of Se per mole of biologically active tRNA. Loss of Se from the modified nucleoside, mnm 5Se 2U, in this tRNA results in concomitant loss of glutamate charging activity suggesting that selenium is essential for interaction of the synthetase and its cognate tRNA.

Role of nuclear genes in expression of a mitochondrial tRNA gene in Saccharomyces cerevisiae.

In yeast mitochondria, most of the isoaccepting species of tyrosyl tRNA are coded by a mitochondrial gene, tyrA. A particular isoaccepting species is coded by a second mitochondrial gene, tyrB. This gene is not expressed in certain strains of yeast which show no deficient phenotype. Genetic crosses between strains expressing or not expressing the tyrB gene demonstrate that expression is controlled by specific nuclear genes and that a mutation of the tyrA gene can be bypassed when the tyrB gene is operative.