T-box riboswitches control transcription of downstream genes through the tRNA-binding formation of terminator or antiterminator structures. Previously reported T-boxes were described as single-specificity riboswitches that can bind specific tRNA anticodons through codon–anticodon interactions with the nucleotide triplet of their specifier loop (SL). However, the possibility that T-boxes might exhibit specificity beyond a single tRNA had been overlooked. In Clostridium acetobutylicum , the T-box that regulates the operon for the essential tRNA-dependent transamidation pathway harbors a SL with two potential overlapping codon positions for tRNA Asn and tRNA Glu . To test its specificity, we performed extensive mutagenic, biochemical, and chemical probing analyses. Surprisingly, both tRNAs can efficiently bind the SL in vitro and in vivo. The dual specificity of the T-box is allowed by a single base shift on the SL from one overlapping codon to the next. This feature allows the riboswitch to sense two tRNAs and balance the biosynthesis of two amino acids. Detailed genomic comparisons support our observations and suggest that “flexible” T-box riboswitches are widespread among bacteria, and, moreover, their specificity is dictated by the metabolic interconnection of the pathways under control. Taken together, our results support the notion of a genome-dependent codon ambiguity of the SLs. Furthermore, the existence of two overlapping codons imposes a unique example of tRNA-dependent regulation at the transcriptional level.

The mechanism of action of chloramphenicol in inhibiting peptide bond formation has been examined with the aim of discovering whether chloramphenicol brings about conformational changes in the peptidyltransferase domain, its target locus on the ribosome. These conformational changes have been sought as changes in the catalytic rate constant of peptidyltransferase. A detailed kinetic analysis of the inhibition of the puromycin reaction in a system derived from Escherichia coli [Kalpaxis et al. (1986) Eur. J. Biochem. 151,267-2711 has been carried out. There is an initial phase of competitive inhibition (Ki = 0.7 pM) in which the double-reciprocal plots are linear. This phase is observed at concentrations of chloramphenicol up to about 3.0 pM (4.3 Ki). By increasing the concentration of the inhibitor the kinetics change and the inhibition becomes no longer of the competitive type. These results are obtained when the inhibitor is added simultaneously with the substrate (puromycin). Preincubation with the inhibitor before the addition of puromycin gives hyperbolic double-reciprocal plots at inhibitor concentrations around the Ki. After preincubation with the inhibitor at concentrations above the Ki (3 -100 Ki) the double-reciprocal plots are linear again and indicate complete, mixed non-competitive inhibition.Analogous behaviour is observed with thiamphenicol (Ki = 0.45 pM) and tevenel (Ki = 1.7 pM). It is proposed that initially chloramphenicol and its two analogs interact with puromycin at a ribosomal locus (peptidyltransferase domain) in a mutually exclusive binding mode (competitive kinetics). Soon after this initial interaction, the antibiotic induces conformational changes to the peptidyltransferase domain so that puromycin is accepted and peptide bonds are still formed but with a lower catalytic rate constant. At this latter state, the ribosome can accept both the inhibitor and the substrate (puromycin) but then, if the concentration of the inhibitor is sufficiently high, peptide bonds are not formed (complete, linear mixed non-competitive inhibition).

In Staphylococcus aureus, a T-box riboswitch exists upstream of the glyS gene to regulate transcription of the sole glycyl-tRNA synthetase, which aminoacylates five tRNA Gly isoacceptors bearing GCC or UCC anticodons. Subsequently, the glycylated tRNAs serve as substrates for decoding glycine codons during translation, and also as glycine donors for exoribosomal synthesis of pentaglycine peptides during cell wall formation. Probing of the predicted T-box structure revealed a long stem I, lacking features previously described for similar T-boxes. Moreover, the antiterminator stem includes a 42-nt long intervening sequence, which is staphylococci-specific. Finally, the terminator conformation adopts a rigid two-stem structure, where the intervening sequence forms the first stem followed by the second stem, which includes the more conserved residues. Interestingly, all five tRNA Gly isoacceptors interact with S. aureus glyS T-box with different binding affinities and they all induce transcription readthrough at different levels. The ability of both GCC and UCC anticodons to interact with the specifier loop indicates ambiguity during the specifier triplet reading, similar to the unconventional reading of glycine codons during protein synthesis. The S. aureus glyS T-box structure is consistent with the recent crystallographic and NMR studies, despite apparent differences, and highlights the phylogenetic variability of T-boxes when studied in a genome-dependent context. Our data suggest that the S. aureus glyS T-box exhibits differential tRNA selectivity, which possibly contributes toward the regulation and synchronization of ribosomal and exoribosomal peptide synthesis, two essential but metabolically unrelated pathways.

No abstract

No abstract

Ribonuclease P (RNase P) from Dictyostelium discoideum has been purified 470-fold. D. discoideum RNase P cleaves the precursor to Schizosaccharomyces pombe suppressor tRNAS" at the same site as S. pombe RNase P, producing the mature 5' end of tRNAS". pH and temperature optima for enzyme activity are 7.6 and 37"C, respectively. The enzyme shows optimal activity in the presence of 5 mM MgC1, and 10 niM NH,Cl or 5 mM KCl. The apparent K,,, for the S. pombe tRNA precursor derived from the sups1 tRNAFer gene is 240 nM, and the apparent V,,, is 3.6 pmollmin. Inhibition of D. discoideum RNase P by proteinase K and micrococcal nuclease strongly indicates that the activity requires both protein and RNA components. In cesium sulfate density gradients, the enzyme has a buoyant density of 1.23 g/ml, indicating a low RNNprotein ratio for the holoenzyme. Fax: +30 61 997690.Abbreviation. RNase P, ribonuclease P.

RNase P from Schizosaccharomyces pombe has been purified over 2000-fold. The apparent K,,, for two S. pombe tRNA precursors derived from the supSI and sup3-e tRNAScr genes is 20 nM; the apparent V,, is 2.5 nM/min (supS1) and 1.1 nM/min (supjl-e). Processing studies with precursors of other mutants show that the structures of the acceptor stem and anticodon/intron loop of tRNA are crucial for S. pombe RNase P action.

Ribonuclease P from the fission yeast Schizosaccharomyces pombe has been purified to apparent homogeneity. A purification of 23 000-fold was achieved by four fractionation steps with DEAEcellulose chromatography, phosphocellulose chromatography, glycerol-gradient fractionation and finally tRNA-affinity chromatography. A 100-kDa protein was present in the most pure preparations in amounts approximately stoichiometric with the previously identified RNA components of the enzyme, K1-RNA and K2-RNA {Krupp, G., Cherayil, B., Frendeway, D., Nishikawa, S. & SOH, D. (1986) EMBO J . 5, 1697-17031. A cross-linking experiment utilizing a 4-thiouridine-substituted precursor tRNA demonstrated that the 1 00-kDa protein interacts with the ribonuclease P substrate in a specific fashion. We therefore conclude that the protein component of S. pombe ribonuclease P is a 100-kDa protein.Ribonuclease P (RNase P) has generated much interest during the past decade because of the catalytic properties of its RNA component. Most attention has focused on the enzymes from Escherichia coli and BaciElus subtilis, where RNase P consists of 90% RNA and the protein component is smaller than the tRNA substrate Altman, 1989;Pace and Smith, 1990;Altman, 1990). In these eubacterial enzymes, the RNA component alone is sufficient to direct 5'-end maturation of tRNA in vitro. In vivo, the protein is essential and is involved in the fine-tuning and acceleration of the catalytic reaction.RNase P activities have also been detected in archaebacteria, eukaryotic nuclei, mitochondria and chloroplasts, but these activities have not been investigated thoroughly. Nonetheless, virtually all RNase P enzymes have been shown to contain an essential RNA component. Despite the presence of RNA, some of these enzymes display striking differences from the eubacteiial enzymes which suggests that the protein components serve a more vital function. A markedly higher protein content is found in enzymes from mitochondria (Morales et al., 1989), eukaryotic nuclei (Bartkiewicz et al., 1989;Kline et al., 1981 ;Lee and Engelke, 1989), chloroplasts (Wang et a]., 1988) and an archaebacterium (Darr et al., 1990). In yeast mitochondria1 RNase P, fragments of the RNA component totaling one third of the in vivo RNA levels are sufficient to support activity (Morales et al., 1989). In the most extreme instance, it has been speculated that chloroplast RNase P may lack an RNA component altogether (Wang et al., 1988; Darr et al., 1992). Thus, it is not yet clear if the RNA components of all RNase P enzymes are catalytic to the same extent as in E. coli and B. subtilis. It is necessary