T box riboswitches in Actinobacteria: translational regulation via novel tRNA interactions.

The T box riboswitch regulates many amino acid-related genes in Gram-positive bacteria. T box riboswitch-mediated gene regulation was shown previously to occur at the level of transcription attenuation via structural rearrangements in the 5' untranslated (leader) region of the mRNA in response to binding of a specific uncharged tRNA. In this study, a novel group of isoleucyl-tRNA synthetase gene (ileS) T box leader sequences found in organisms of the phylum Actinobacteria was investigated. The Stem I domains of these RNAs lack several highly conserved elements that are essential for interaction with the tRNA ligand in other T box RNAs. Many of these RNAs were predicted to regulate gene expression at the level of translation initiation through tRNA-dependent stabilization of a helix that sequesters a sequence complementary to the Shine-Dalgarno (SD) sequence, thus freeing the SD sequence for ribosome binding and translation initiation. We demonstrated specific binding to the cognate tRNA(Ile) and tRNA(Ile)-dependent structural rearrangements consistent with regulation at the level of translation initiation, providing the first biochemical demonstration, to our knowledge, of translational regulation in a T box riboswitch.

MupB, a new high-level mupirocin resistance mechanism in Staphylococcus aureus.

Mupirocin is a topical antibiotic used for the treatment of skin infections and the eradication of methicillin-resistant Staphylococcus aureus carriage. It inhibits bacterial protein synthesis by interfering with isoleucyl-tRNA synthetase activity. High-level mupirocin resistance (MIC of ≥ 512 µg/ml) is mediated by the expression of mupA (ileS2), which encodes an alternate isoleucyl-tRNA synthetase. In this study, we describe high-level mupirocin resistance mediated by a novel locus, mupB. The mupB gene (3,102 bp) shares 65.5% sequence identity with mupA but only 45.5% identity with ileS. The deduced MupB protein shares 58.1% identity (72.3% similarity) and 25.4% identity (41.8% similarity) with MupA and IleS, respectively. Despite this limited homology, MupB contains conserved motifs found in class I tRNA synthetases. Attempts to transfer high-level mupirocin resistance via conjugation or transformation (using plasmid extracts from an mupB-containing strain) were unsuccessful. However, by cloning the mupB gene into a shuttle vector, it was possible to transfer the resistance phenotype to susceptible S. aureus by electroporation, proving that mupB was responsible for the high-level mupirocin resistance. Further studies need to be done to determine the prevalence of mupB and to understand risk factors and outcomes associated with resistance mediated by this gene.

Interaction between human tRNA synthetases involves repeated sequence elements.

Aminoacyl-tRNA synthetases (tRNA synthetases) of higher eukaryotes form a multiprotein complex. Sequence elements that are responsible for the protein assembly were searched by using a yeast two-hybrid system. Human cytoplasmic isoleucyl-tRNA synthetase is a component of the multi-tRNA synthetase complex and it contains a unique C-terminal appendix. This part of the protein was used as bait to identify an interacting protein from a HeLa cDNA library. The selected sequence represented the internal 317 amino acids of human bifunctional (glutamyl- and prolyl-) tRNA synthetase, which is also known to be a component of the complex. Both the C-terminal appendix of the isoleucyl-tRNA synthetase and the internal region of bifunctional tRNA synthetase comprise repeating sequence units, two repeats of about 90 amino acids, and three repeats of 57 amino acids, respectively. Each repeated motif of the two proteins was responsible for the interaction, but the stronger interaction was shown by the native structures containing multiple motifs. Interestingly, the N-terminal extension of human glycyl-tRNA synthetase containing a single motif homologous to those in the bifunctional tRNA synthetase also interacted with the C-terminal motif of the isoleucyl-tRNA synthetase although the enzyme is not a component of the complex. The data indicate that the multiplicity of the binding motif in the tRNA synthetases is necessary for enhancing the interaction strength and may be one of the determining factors for the tRNA synthetases to be involved in the formation of the multi-tRNA synthetase complex.

A eubacterial Mycobacterium tuberculosis tRNA synthetase is eukaryote-like and resistant to a eubacterial-specific antisynthetase drug.

We report here the cloning and primary structure of Mycobacterium tuberculosis isoleucyl-tRNA synthetase. The predicted 1035-amino acid protein is significantly more similar in sequence to eukaryote cytoplasmic than to other eubacterial isoleucyl-tRNA synthetases. This similarity correlates with the enzyme being resistant to pseudomonic acid A, a potent inhibitor of Escherichia coli and other eubacterial isoleucyl-tRNA synthetases, but not of eukaryote cytoplasmic enzymes. Consistent with its eukaryote-like features, and unlike E. coli isoleucyl-tRNA synthetase, the M. tuberculosis enzyme charged yeast isoleucine tRNA. In spite of these eukaryote-like features, M. tuberculosis isoleucyl-tRNA synthetase exhibited highly specific cross-species aminoacylation, as demonstrated by its ability to complement isoleucyl-tRNA synthetase-deficient mutants of E. coli. When introduced into a pseudomonic acid-sensitive wild-type strain of E. coli, the M. tuberculosis enzyme conferred trans-dominant resistance to the drug. The results demonstrate that the sequence of a tRNA synthetase could have predictive value with respect to the interaction of that synthetase with a specific inhibitor. The results also demonstrate that mobilization of a pathogen's gene for a drug-resistant protein target can spread resistance to other, normally drug-sensitive pathogens infecting the same host.

Analysis and toxic overexpression in Escherichia coli of a staphylococcal gene encoding isoleucyl-tRNA synthetase.

We have cloned and sequenced the Staphylococcus aureus Oxford ileS gene which encodes isoleucyl-tRNA synthetase (Ile-RS), the target for the antibiotic mupirocin. The gene was identified by hybridisation to oligodeoxyribonucleotide probes derived from internal Ile-RS amino acid (aa) sequences. The 2754-bp open reading frame encodes a 918-aa protein of 105 kDa which is homologous to other known Ile-RS from Gram- bacteria, archaebacteria, yeast and protozoa. Motifs which have been implicated in the functioning of the active site are strongly conserved. The gene was engineered for high-level expression in Escherichia coli. Ile-RS overproduction was toxic to the E. coli host, the magnitude of its observed effects being strain-dependent.

Evidence for cAMP-mediated control of isoleucyl-tRNA synthetase formation in Escherichia coli K-12.

The ability of cAMP to inhibit isoleucyl-tRNA synthetase (IRS) formation has been demonstrated in wild type K-12 Escherichia coli and two adenyl-cyclase (cya) mutants. cAMP appeared not to have any effect on either the valyl- or arginyl-tRNA synthetase (VRS and ARS respectively). Addition of cAMP led to a reduction in rate of IRS synthesis but not VRS or ARS. Furthermore, derepression of IRS and VRS by isoleucine limitation was completely prevented by cAMP.

Synthesis of the isoleucyl- and valyl-tRNA synthetases and the isoleucine-valine biosynthetic enzymes in a threonine deaminase regulatory mutant of Escherichia coli K-12.

A mutation in the structural gene for threonine deaminase, ilvA538 , results in lower than normal levels of the isoleucyl, valyl- and leucyl-tRNA synthetases. Moreover, this regulatory mutation decreases the level of expression of the ilv biosynthetic operons and renders their expression non-responsive to limitations of the branched-chain amino acids. In this paper, we present in vitro evidence for the inhibition of isoleucyl- and valyl-tRNA synthetase activity by threonine deaminase and 2-ketobutyrate, the product of the threonine deaminase reaction, through the formation of a high molecular weight complex of the three molecules. Based on these results, we propose a model to explain the regulation of the isoleucyl- and valyt -tRNA synthetases in which transient inhibition of the synthetase enzyme activities by threonine deaminase and 2-ketobutyrate increases the expression of ileS and valS , the structural genes for isoleucyl- and valyt -tRNA synthetase, respectively. Further, the results suggest that the hyperattenuated expression of the ilv biosynthetic operons is due to an increased rate of complex formation of valyl and isoleucyl-tRNA synthetases and the altered form of threonine deaminase of the ilvA538 mutant strain.

Ribosomal protein S20 purified under mild conditions almost completely inhibits its own translation.

The efficiency of ribosomal protein S20 to act as repressor of its own synthesis in an in vitro system was found to depend greatly on the procedures employed to purify this protein. Whilst conventionally purified r-protein S20 inhibited its own synthesis by some 30%, up to 90% inhibition was observed if "milder" purification conditions were used. Evidence is presented that the latter preparation shows also a higher binding affinity to 16S rRNA.

Factors modulating transcription and translation in vitro of ribosomal protein S20 and isoleucyl-tRNA synthetase from Escherichia coli.

The DNA-dependent protein-synthesizing system developed by Zubay [Zubay, G. (1973) Annu. Rev. Genet. 7, 267--287] was optimized for the transcription and translation of genes from the 0.5-min region of the Escherichia coli chromosome carried by transducing lambda phages. The E. coli gene products synthesized were isoleucyl tRNA synthetase, ribosomal protein S20, dihydrodipicolinic acid reductase and (possibly) the two subunits carbamoyl-phosphate synthetase. Formation of ribosomal protein S20 is specifically stimulated by the addition of 16-S rRNA and not by 5-S or 23-S rRNA. 16-S rRNA increases the rate of S20 synthesis, the final yield of product depends on the duration of persistence of the RNA added. Addition of 16-S rRNA to the separate transcription and translation systems showed that it is the translation of the S20 mRNA which is enhanced. Furthermore, S20 synthesis is stimulated more than fourfold when concomitant synthesis of rRNA occurs from a plasmid carrying an rrn transcriptional unit. The results described are explained in terms of a model which suggests that ribosomal protein S20 feedback inhibits its synthesis at the translational level and that removal of S20 into ribosomal assembly (i.e. binding to 16-S rRNA) releases inhibition. The model postulates a direct link between synthesis of ribosomal RNA and ribosomal protein and between the rates of ribosomal assembly and ribosomal protein synthesis. The stimulatory effect of guanosine 3'-diphosphate 5'-diphosphate on isoleucyl-tRNA synthetase formation and its inhibition of the synthesis of ribosomal protein S20 in vitro occurs at the level of transcription. Its relevance in vivo, however, remains to be demonstrated. Formation of isoleucyl-tRNA synthetase in vitro is not influenced either by the addition of a surplus of purified enzyme nor by the limitation of protein synthesis by the addition of anti-(isoleucyl-tRNA synthetase) serum. There is no evidence, therefore, that isoleucyl-tRNA synthetase is autogenously regulated.

[Regulation of the biosynthesis of branched aminoacyl tRNA synthetases in Bacillus cereus T]. / Régulation de la biosynthèse des aminoacyl tRNA synthétases des acides aminés branchés chez Bacillus cereus T.

The regulation of the biosynthesis of isoleucyl-, valyl-, and leucyl-tRNA synthetases was studied with an isoleucine-valine requiring mutant of Bacillus cereus T. It was shown that valyl-tRNA synthetase regulation is under multivalent control involving both valine and isoleucine. The isoleucyl- and leucyl-tRNA synthetases are repressed by the respective cognate amino acid. When two amino acids were removed from the culture medium, derepression of the two corresponding aminoacyl tRNA synthetases was expected, but only one appears. With a threonine deaminase constitutive mutant, it was demonstrated that the derepression mecanism of the synthetase was correlated with the intracellular level of the ilv A gene product. These results are in good agreement with the model proposed by several authors. In this model, threonine deaminase, or some form of this enzyme, is involved in a positive control of the regulation of the branched-chain aminoacyltransfer ribonucleic acid synthetases.

ilvU, a locus in Escherichia coli affecting the derepression of isoleucyl-tRNA synthetase and the RPC-5 chromatographic profiles of tRNAIle and tRNAVal.

A mutation in the ilvU locus of Escherichia coli has led to a complex phenotype that included resistance to thiaisoleucine, a loss of derepressibility of isoleucyl tRNA synthetase, and an alteration of the RPC-5 chromatographic profile of the branched-chain aminoacyl-tRNA's. The alterations were manifest in an increase in the amount of Species 2 of both tRNAIle and tRNAVal at the expense of Species 1. A similar alteration, but independent of (and additive to) that caused by the ilvU mutation, was observed upon limitation of either isoleucine or valine. The shift in profile caused by limitation was also independent of the reduced growth rate or the derepression of the isoleucine and valine biosynthetic enzymes that also result from limitation. During chloramphenicol treatment nearly all tRNAIle and tRNAVal formed appears as species 2. Upon recovery from chloramphenicol, Species 2 of both acceptors are converted to Species 1. It is proposed that the ilvU product not only allows derepression of isoleucyl-tRNA synthetase but also retards the conversion of tRNA2Ile to tRNA1Ile and that of tRNA2Val to tRNA1Val. The mutated ilvU loci abolish the derepression and are more efficient in retarding the conversion.

Transient rates of synthesis of five amionacyl-transfer ribonucleic acid synthetases during a shift-up of Escherichia coli.

The steady-state levels of a number of aminoacyl-transfer ribonucleic acid synthetases are known to be positively correlated with growth rate in Escherichia coli. To describe the regulation of these enzymes during a nutritional shift-up, use was made of the recent identification of polypeptide chains of several synthetases in whole cell lysates resolved by the O'Farrell two-dimensional gel system. A culture growing in acetate minimal medium was shifted to glucose-rich medium and pulse labeled with [3H]leucine and [3H]isoleucine for 30- or 6-s intervals during the 20 min after the shift. After mixing with a uniformly [35S]sulfate-labeled reference culture, the samples were subjected to two-dimensional gel electrophoresis. The 3H/35S ratio in the resolved synthetase polypeptides provided an accurate estimation of their transient rates of synthesis. Five aminoacyl-transfer ribonucleic acid synthetases (those for argnine, glycine, isoleucine, phenylalanine, and valine) exhibited an increase in formation within 30 to 90 s after the shift-up. The magnitude of the increases corresponded to the final steady-state values and were reached within 2 to 3 min. The addition to rifampin revealed that the increase in the differential rate of valyl-transfer ribonucleic acid synthetase formation was the result of increased messenger ribonucleic acid transcription and not of a relaxation of some translation restriction.