RESUMEN
The expression of the gene thrS encoding threonyl-tRNA synthetase is under the control of two apparently different regulatory loops: translational feedback regulation and growth rate-dependent control. The translational feedback regulation is due to the binding of threonyl-tRNA synthetase to a site located in the leader RNA of thrS, upstream of the initiation codon, which mimics the anticodon stem and loop of tRNA(Thr). This binding competes with that of the ribosome and thus inhibits translation initiation. Here, we investigate the mechanism of growth rate-dependent control, i.e. the mechanism by which the synthetase accumulates at high growth rates. We show that growth rate-dependent control acts at the level of translation and requires feedback regulation since mutations that abolish feedback regulation also abolish growth rate-dependent control. We also show that tRNA(Thr), which accumulates at high growth rates, is one of the effectors of growth rate-dependent control since its accumulation can cause derepression independently of growth rate. We show that this tRNA(Thr)-dependent derepression is also dependent on feedback regulation since mutations which abolish feedback also prevent derepression. Based on these results and previous data concerning the mechanism of translational feedback regulation, we propose that threonyl-tRNA synthetase growth rate-dependent control is the consequence of the accumulation at high growth rates of two effectors, the ribosome and tRNA(Thr). We also study the growth rate-dependence of the steady state level of thrS mRNA and show that the steady state level of thrS mRNA increases at high growth rates. This increase is dependent on the translational feedback regulation and can also be detected, independently of growth rate, when thrS mRNA translation is derepressed. Consistently with the model of growth rate-dependent control above, we propose that at high growth rates, the mRNA is well translated and thus stabilised and that, at low growth rates, because of its low translation, thrS mRNA is rapidly degraded.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , ARN Mensajero/biosíntesis , Treonina-ARNt Ligasa/genética , Secuencia de Bases , Escherichia coli/crecimiento & desarrollo , Retroalimentación , Regulación Enzimológica de la Expresión Génica/fisiología , Datos de Secuencia Molecular , Mutación , Biosíntesis de Proteínas/fisiología , ARN Bacteriano/biosíntesis , ARN Mensajero/metabolismo , ARN de Transferencia de Treonina/fisiología , Proteínas Recombinantes de Fusión , Treonina-ARNt Ligasa/metabolismo , Valina-ARNt Ligasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
The alpha and beta subunits of phenylalanyl-tRNA synthetase are encoded by the pheS and pheT genes, respectively. These genes are clustered closely together with the genes for threonyl-tRNA synthetase (thrS) and translation initiation factor IF3 (infC); the gene order is thrS infC pheS pheT. We have used two methods to study the transcription pattern within this cluster. The first was the in vitro transcription of DNA restriction fragments with purified RNA polymerase, followed by fractionation of the RNA products by polyacrylamide gel electrophoresis. The second method was the mapping of promoters by means of the "abortive initiation" reaction of McClure and co-workers. This procedure consists of the incubation of RNA polymerase with DNA restriction fragments plus one nucleoside monophosphate and one [alpha-32P]nucleoside triphosphate; the polymerase synthesizes dinucleotide products of known sequence at promoter sites in the DNA. We found that transcription initiated at an internal site within infC (designated P1), and at two promoter sites between infC and pheS (designated P2 and P3). Transcription terminated at two sites about 200 nucleotides apart, located just before pheS. The initiation and termination signals were arranged so as to yield a nested set of overlapping transcripts. At the P1 promoter, transcription initiated with G-C, at P2 with A-C and sometimes A-G, and at P3 with G-U. Promoter activity was also found in a 3000-base interval that includes the start of the thrS gene; eight or nine transcripts (not mapped in detail) were observed, which started with at least four different dinucleotides. All major initiation sites in the gene cluster represented purine starts, although some pyrimidine initiation was observed in trace amounts. No promoter activity was found between pheS and pheT with either of the two techniques; this observation supports the conclusion that these genes are co-transcribed. No evidence was found for any promoter between the termination sites and the beginning of the pheS gene. It is suggested that one of the terminators is an attenuation site controlling the extension of transcription into pheS and pheT. Attenuation may explain the observed regulation of phenylalanyl-tRNA synthetase by the amino acid supply.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Escherichia coli/genética , Genes Bacterianos , Factores de Iniciación de Péptidos/genética , Transcripción Genética , Autorradiografía , Secuencia de Bases , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN/genética , Operón , Fenilalanina-ARNt Ligasa/genética , Factor 3 Procariótico de Iniciación , Treonina-ARNt Ligasa/genéticaRESUMEN
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.
Asunto(s)
Escherichia coli/genética , Genes Bacterianos , Aminoacil-ARN de Transferencia/genética , Fraccionamiento Químico , Cromatografía DEAE-Celulosa , Codón/genética , Regulación de la Expresión GénicaRESUMEN
The organization of seven genes located at about 38 min on the genetic map of Escherichia coli was examined; these genes included pheS and pheT, which code for the alpha and beta subunits of phenylalanyl-transfer ribonucleic acid synthetase, and thrS, the structural gene for threonyl-transfer ribonucleic acid synthetase. Deletion mutants were isolated from an F-prime-containing merodiploid strain and were characterized genetically. Seventeen different kinds of deletions extending into pheS of pheT were identified. These deletions unambiguously defined the gene order as aroD pps himA pheT pheS thrS pfkB. Mutants with deletions covering either pheS or pheT, but not both, were analyzed further by assay of phenylalanyl-transfer ribonucleic acid synthetase. The phenotype of the mutants with a deletion from pfkB through pheS was anomalous; although the pheT gene was apparently still present, its product, the beta subunit, was much reduced in activity.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Escherichia coli/genética , Fenilalanina-ARNt Ligasa/genética , Mapeo Cromosómico , Escherichia coli/enzimología , Mutación , Fenilalanina-ARNt Ligasa/metabolismo , Transcripción GenéticaAsunto(s)
Colifagos , ARN de Transferencia , Mapeo Cromosómico , Mutación , ARN Viral , Recombinación GenéticaRESUMEN
The phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli is a tetramer that contains two different kinds of polypeptide chains. To locate the genes for the two polypeptides, we analyzed temperature-sensitive mutants with defective phenylalanyl-transfer ribonucleic acid synthetases to see which subunit was altered. The method was in vitro complementation; mutant cell extracts were mixed with purified separated alpha or beta subunits of the wild-type enzyme to generate an active hybrid enzyme. With three mutants, enzyme activity appeared when alpha was added, but not when beta was added: these are, therefore, assumed to carry lesions in the gene for the alpha subunit. Two other mutants gave the opposite response and are presumably beta mutants. Enzyme activity is also generated when alpha and beta mutant extracts are mixed, but not when two alpha or two beta mutant extracts are mixed. The inactive mutant enzymes appear to be dissociated, as judged by their sedimentation in sucrose density gradients, but the dissociation may be only partial. The active enzyme generated by complementation occurred in two forms, one that resembled the native wild-type enzyme and one that sedimented more slowly. Both alpha and beta mutants are capable of generating the native form, although alpha mutants require prior urea denaturation of the defective enzyme. With the mutants thus characterized, the genes for the alpha and beta subunits (designated pheS and heT, respectively) were mapped. The gene order, as determined by transduction is aroD-pps-pheT-pheS. The pheS and pheT genes are close together and may be immediately adjacent.