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1.
RNA ; 7(2): 242-53, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11233981

RESUMO

Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.


Assuntos
Bacillus subtilis/genética , Endorribonucleases/genética , Nucleotidiltransferases/genética , Precursores de RNA/genética , RNA Bacteriano/genética , RNA Ribossômico 5S/genética , Ribossomos/genética , Sequência de Aminoácidos , Pareamento de Bases , Sequência de Bases , Northern Blotting , Núcleo Celular/genética , Núcleo Celular/metabolismo , Sobrevivência Celular/fisiologia , Primers do DNA/química , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Processamento Pós-Transcricional do RNA , RNA Ribossômico 5S/química , Homologia de Sequência de Aminoácidos
2.
Biochimie ; 82(12): 1091-8, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11120350

RESUMO

The Escherichia coli protein synthesis initiation factor IF2 is a member of the large family of G-proteins. Along with translational elongation factors EF-Tu and EF-G and translational release factor RF-3, IF2 belongs to the subgroup of G-proteins that are part of the prokaryotic translational apparatus. The roles of IF2 and EF-Tu are similar: both promote binding of an aminoacyl-tRNA to the ribosome and hydrolyze GTP. In order to investigate the differences and similarities between EF-Tu and IF2 we have created point mutations in the G-domain of IF2, Thr445 to Cys, Ile500 to Cys, and the double mutation. Threonine 445 (X1), which corresponds to cysteine 81 in EF-Tu, is well conserved in the DX1X2GH consensus sequence that has been proposed to interact with GTP. The NKXD motif, in which X is isoleucine 500 in IF2, corresponds to cysteine 137 in EF-Tu, and is responsible for the binding of the guanine ring. The recombinant mutant proteins were expressed and tested in vivo for their ability to sustain growth of an Escherichia coli strain lacking the chromosomal copy of the infB gene coding for IF2. All mutated proteins resulted in cell viability when grown at 42 degrees C or 37 degrees C. However, Thr445 to Cys mutant showed a significant decrease in the growth rate at 25 degrees C. The mutant proteins were overexpressed and purified. As observed in vivo, a reduced activity at low temperature was measured when carrying out in vitro ribosome dependent GTPase and stimulation of ribosomal fMet-tRNAfMet binding.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Mutação , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Sequência de Aminoácidos , Divisão Celular , Sequência Conservada , Fator de Iniciação 5 em Eucariotos , GTP Fosfo-Hidrolases/metabolismo , Teste de Complementação Genética , Guanosina Trifosfato/metabolismo , Isoleucina , Mutação Puntual , Conformação Proteica , Estrutura Terciária de Proteína , Temperatura , Treonina
3.
J Biol Chem ; 274(10): 6074-9, 1999 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-10037688

RESUMO

We have recently shown that the Escherichia coli initiation factor 2 (IF2) G-domain mutants V400G and H448E do not support cell survival and have a strong negative effect on growth even in the presence of wild-type IF2. We have isolated both mutant proteins and performed an in vitro study of their main functions. The affinity of both mutant proteins for GTP is almost unchanged compared with wild-type IF2. However, the uncoupled GTPase activity of the V400G and H448E mutants is severely impaired, the Vmax values being 11- and 40-fold lower, respectively. Both mutant forms promoted fMet-tRNAfMet binding to 70 S ribosomes with similar efficiencies and were as sensitive to competitive inhibition by GDP as wild-type IF2. Formation of the first peptide bond, as measured by the puromycin reaction, was completely inhibited in the presence of the H448E mutant but still significant in the case of the V400G mutant. Sucrose density gradient centrifugation revealed that, in contrast to wild-type IF2, both mutant proteins stay blocked on the ribosome after formation of the 70 S initiation complex. This probably explains their dominant negative effect in vivo. Our results underline the importance of GTP hydrolysis for the recycling of IF2.


Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Mutação , Fatores de Iniciação de Peptídeos/genética , Escherichia coli/crescimento & desenvolvimento , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Hidrólise , Fatores de Iniciação de Peptídeos/metabolismo , Fator de Iniciação 2 em Procariotos
4.
Annu Rev Genet ; 33: 193-227, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10690408

RESUMO

The stability of mRNA in prokaryotes depends on multiple factors and it has not yet been possible to describe the process of mRNA degradation in terms of a unique pathway. However, important advances have been made in the past 10 years with the characterization of the cis-acting RNA elements and the trans-acting cellular proteins that control mRNA decay. The trans-acting proteins are mainly four nucleases, two endo- (RNase E and RNase III) and two exonucleases (PNPase and RNase II), and poly(A) polymerase. RNase E and PNPase are found in a multienzyme complex called the degradosome. In addition to the host nucleases, phage T4 encodes a specific endonuclease called RegB. The cis-acting elements that protect mRNA from degradation are stable stem-loops at the 5' end of the transcript and terminators or REP sequences at their 3' end. The rate-limiting step in mRNA decay is usually an initial endonucleolytic cleavage that often occurs at the 5' extremity. This initial step is followed by directional 3' to 5' degradation by the two exonucleases. Several examples, reviewed here, indicate that mRNA degradation is an important step at which gene expression can be controlled. This regulation can be either global, as in the case of growth rate-dependent control, or specific, in response to changes in the environmental conditions.


Assuntos
Bactérias/genética , Bacteriófagos/genética , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Transcrição Gênica
5.
Nucleic Acids Res ; 26(23): 5379-87, 1998 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-9826762

RESUMO

The Bacillus subtilis thrS gene is a member of the T-box gene family in Gram-positive organisms whose expression is regulated by a tRNA-mediated transcriptional antitermination mechanism involving a direct tRNA:mRNA interaction. The complex leader sequences of these genes share only short stretches of primary sequence homology, but a common secondary structure has been proposed by comparing the leaders of many genes of this family. The proposed mechanism forthe tRNA:mRNA interaction depends heavily on the secondary structure model, but is so far only supported by genetic evidence. We have studied the structure of the B.subtilis thrS leader in solution, in protection experiments using both chemical and enzymatic probes. The thrS leader structure was also probed in vivo using dimethylsulphate and the in vitro and in vivo data are in good accordance. We have organized the thrS leader into three major domains comprising six separate stem-loops. All but one of the short sequences conserved in this gene family are present in loop structures. The ACC specifier codon proposed to interact with the tRNAThrGGUisoacceptor is present in a bulge and probably exists in a stacking conformation. The proposed antiterminator structure is not visible in transcripts containing the terminator, but was probed using a transcript with the 3'-half of the terminator deleted and its folding appears consistent with the regulatory model. The leader sequences, and in particular the specifier domains, of the other genes of this family can be folded similarly to the experimentally solved thrS structure.


Assuntos
Bacillus subtilis/genética , Conformação de Ácido Nucleico , Treonina-tRNA Ligase/química , Treonina-tRNA Ligase/genética , Regiões 5' não Traduzidas/química , Regiões 5' não Traduzidas/efeitos dos fármacos , Regiões 5' não Traduzidas/genética , Alquilantes/farmacologia , Bacillus subtilis/enzimologia , Sequência de Bases/efeitos dos fármacos , Sequência Conservada , Modelos Químicos , Dados de Sequência Molecular , Mutagênicos/farmacologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Sondas RNA , RNA Bacteriano/química , RNA Bacteriano/efeitos dos fármacos , Ésteres do Ácido Sulfúrico/farmacologia
6.
Proc Natl Acad Sci U S A ; 95(5): 2564-7, 1998 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-9482926

RESUMO

The ribosomal protein S15 from Escherichia coli binds to a pseudoknot in its own messenger. This interaction is an essential step in the mechanism of S15 translational autoregulation. In a previous study, a recognition determinant for S15 autoregulation, involving a U.G wobble pair, was located in the center of stem I of the pseudoknot. In this study, an extensive mutagenesis analysis has been conducted in and around this U.G pair by comparing the effects of these mutations on the expression level of S15. The results show that the U.G wobble pair cannot be substituted by A.G, C.A, A.C, G.U, or C.G without loss of the autocontrol. In addition, the base pair C.G, adjacent to the 5' side of U, cannot be flipped or changed to another complementary base pair without also inducing derepression of translation. A unique motif, made of only two adjacent base pairs, U.G/C.G, is essential for S15 autoregulation and is presumably involved in direct recognition by the S15 protein.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/biossíntese , Composição de Bases , Sequência de Bases , Sítios de Ligação , Guanina , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Ribossômicas/química , Uracila , beta-Galactosidase/biossíntese
7.
Eur J Biochem ; 246(2): 291-300, 1997 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-9208917

RESUMO

A 6-kb DNA fragment from an extreme thermophile, Thermus thermophilus, carrying the genes for cytochrome oxidase ba3 subunit I (cbaA) and the ribosomal protein S15 (rpsO) was cloned into Escherichia coli. The gene rpsO was sequenced. The deduced amino acid sequence exhibits 59% identity to the corresponding protein from E. coli. Expression of rpsO in E. coli requires the use of a fully repressed inducible promoter because S15 from T. thermophilus is toxic for E. coli cells. When purified without denaturation from either overproducing E. coli strain or from T. thermophilus ribosomes, the S15 protein is stable and binds a cloned T. thermophilus 16S rRNA fragment (nucleotides 559-753), with low identical dissociation constants (2.5 nM), thus demonstrating that the thermophilic protein folds correctly in a mesophilic bacterium. The rRNA fragment bound corresponds in position and structure to the 16S rRNA fragment of E. coli. A similar high affinity was also found for the binding of S15 from T. thermophilus or E. coli to the corresponding E. coli 16S rRNA fragment, whereas a slightly lower affinity was observed in binding experiments between E. coli S15 and T. thermophilus 16S rRNA fragment. These results suggest that S15 from T. thermophilus recognizes similar determinants in both rRNA fragments. Competition experiments support this conclusion.


Assuntos
Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Thermus thermophilus/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Escherichia coli/genética , Dados de Sequência Molecular , Plasmídeos , RNA Ribossômico 16S/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Homologia de Sequência de Aminoácidos
8.
J Mol Biol ; 268(2): 235-42, 1997 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-9159466

RESUMO

We have recently reported that processing occurs in the untranslated leader region of several members of a family of Gram-positive genes regulated by tRNA-mediated antitermination. We showed that cleavage at this site plays an important role in the induction of Bacillus subtilis thrS gene expression, following threonine starvation, by stabilising the downstream mRNA. Here we show that, when transferred on a plasmid, processing of the B. subtilis thrS leader can occur at the same site in Escherichia coli. Cleavage at this site is dependent on the E. coli endoribonuclease E, both in vivo and in vitro, suggesting that a functional homologue of RNase E is responsible for thrS processing in B. subtilis.


Assuntos
Bacillus subtilis/genética , Endorribonucleases/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Treonina-tRNA Ligase/genética , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , Sequências Reguladoras de Ácido Nucleico
9.
J Bacteriol ; 179(8): 2472-8, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9098041

RESUMO

We have sequenced the valyl-tRNA synthetase gene (valS) of Bacillus subtilis and found an open reading frame coding for a protein of 880 amino acids with a molar mass of 101,749. The predicted amino acid sequence shares strong similarity with the valyl-tRNA synthetases from Bacillus stearothermophilus, Lactobacillus casei, and Escherichia coli. Extracts of B. subtilis strains overexpressing the valS gene on a plasmid have increased valyl-tRNA aminoacylation activity. Northern analysis shows that valS is cotranscribed with the folC gene (encoding folyl-polyglutamate synthetase) lying downstream. The 300-bp 5' noncoding region of the gene contains the characteristic regulatory elements, T box, "specifier codon" (GUC), and rho-independant transcription terminator of a gene family in gram-positive bacteria that encodes many aminoacyl-tRNA synthetases and some amino acid biosynthetic enzymes and that is regulated by tRNA-mediated antitermination. We have shown that valS expression is induced by valine limitation and that the specificity of induction can be switched to threonine by changing the GUC (Val) specifier triplet to ACC (Thr). Overexpression of valS from a recombinant plasmid leads to autorepression of a valS-lacZ transcriptional fusion. Like induction by valine starvation, autoregulation of valS depends on the presence of the GUC specifier codon. Disruption of the valS gene was not lethal, suggesting the existence of a second gene, as is the case for both the thrS and the tyrS genes.


Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Valina-tRNA Ligase/genética , Acilação , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Sequência de Bases , Mapeamento Cromossômico , DNA Recombinante , Escherichia coli/genética , Genes Bacterianos/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Peptídeo Sintases/genética , RNA Bacteriano/análise , RNA Bacteriano/química , RNA Mensageiro/análise , RNA Mensageiro/química , RNA de Transferência de Valina/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição Gênica/genética
11.
J Biol Chem ; 271(37): 22321-5, 1996 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-8798391

RESUMO

According to their role in translation, tRNAs specifically interact either with elongation factor Tu (EFTu) or with initiation factor 2 (IF2). We here describe the effects of overproducing EFTu and IF2 on the elongator versus initiator activities of various mutant tRNAMet species in vivo. The data obtained indicate that the selection of a tRNA through one or the other pathway of translation depends on the relative amounts of the translational factors. A moderate overexpression of EFTu is enough to lead to a misappropriation of initiator tRNA in the elongation process, whereas overproduced IF2 allows the initiation of translation to occur with unformylated tRNA species. In addition, we report that a strain devoid of formylase activity can be cured by the overproduction of tRNAMetf. The present study brings additional evidence for the importance of formylation in defining tRNAMetf initiator identity, as well as a possible explanation for the residual growth of bacterial strains lacking a functional formylase gene such as observed in Guillon, J. M., Mechulam, Y., Schmitter, J.-M., Blanquet, S., and Fayat, G. (1992) J. Bacteriol. 174, 4294-4301.


Assuntos
Fator Tu de Elongação de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , RNA de Transferência de Metionina/metabolismo , Clonagem Molecular , Cosmídeos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Amplificação de Genes , Plasmídeos/metabolismo , Fator de Iniciação 2 em Procariotos , Biossíntese de Proteínas , Mapeamento por Restrição
12.
Proc Natl Acad Sci U S A ; 93(14): 6992-7, 1996 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-8692931

RESUMO

The threonyl-tRNA synthetase gene, thrS, is a member of a family of Gram-positive genes that are induced following starvation for the corresponding amino acid by a transcriptional antitermination mechanism involving the cognate uncharged tRNA. Here we show that an additional level of complexity exists in the control of the thrS gene with the mapping of an mRNA processing site just upstream of the transcription terminator in the thrS leader region. The processed RNA is significantly more stable than the full-length transcript. Under nonstarvation conditions, or following starvation for an amino acid other than threonine, the full-length thrS mRNA is more abundant than the processed transcript. However, following starvation for threonine, the thrS mRNA exists primarily in its cleaved form. This can partly be attributed to an increased processing efficiency following threonine starvation, and partly to a further, nonspecific increase in the stability of the processed transcript under starvation conditions. The increased stability of the processed RNA contributes significantly to the levels of functional RNA observed under threonine starvation conditions, previously attributed solely to antitermination. Finally, we show that processing is likely to occur upstream of the terminator in the leader regions of at least four other genes of this family, suggesting a widespread conservation of this phenomenon in their control.


Assuntos
Bacillus subtilis/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , RNA Mensageiro/metabolismo , Treonina-tRNA Ligase/biossíntese , Treonina/metabolismo , Aminoacil-tRNA Sintetases/biossíntese , Bacillus subtilis/genética , Sequência de Bases , Regulação Enzimológica da Expressão Gênica , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Rifampina/farmacologia , Regiões Terminadoras Genéticas , Transcrição Gênica/efeitos dos fármacos , beta-Galactosidase/biossíntese
13.
Biochemistry ; 35(9): 2978-84, 1996 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-8608135

RESUMO

trans-Diamminedichloroplatinum(II) was used to induce reversible cross-links between Escherichia coli initiation factor 2 (IF-2) and fMet-tRNA(f)(Met). Two distinct cross-links between IF-2 and the initiator tRNA were produced. Analysis of the cross-linking regions on both RNA and protein moieties reveals that the T arm of the tRNA is in the proximity of a region of the C-terminal domain of IF-2 (residues Asn611-Arg645). This cross-link is well-correlated with the fact that the C-domain of IF-2 contains the fMet-tRNA binding site and that the cross-linked RNA fragment precisely maps in a region which is protected by IF-2 from chemical modification and enzymatic digestion. Rather unexpectedly, a second cross-link was characterized which involves the anticodon arm of fMet-tRNA(f)(Met) and the N-terminal part of IF-2 (residues Trp215-Arg237).


Assuntos
Cisplatino/farmacologia , Escherichia coli/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , RNA de Transferência de Metionina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Reagentes de Ligações Cruzadas , Eletroforese em Gel de Poliacrilamida , Escherichia coli/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/isolamento & purificação , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Conformação Proteica , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/isolamento & purificação , Especificidade por Substrato
14.
Biochimie ; 78(6): 381-9, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8915527

RESUMO

In this review, we summarize progress on the regulation of the aminoacyl-tRNA synthetase genes in Bacillus subtilis. Most of the genes encoding this set of enzymes in B subtilis are members of a large family of Gram-positive genes and operons controlled by a novel antitermination mechanism that uses their cognate uncharged tRNA as the effector. A subset of these genes is, in addition, likely to be controlled at the level of mRNA processing and degradation. We describe the key experiments leading to these conclusions.


Assuntos
Aminoacil-tRNA Sintetases/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica/genética , Sequência de Bases , Mapeamento Cromossômico , Sequência Conservada , Dados de Sequência Molecular , Processamento Pós-Transcricional do RNA , Análise de Sequência , Transcrição Gênica/genética
16.
Mol Microbiol ; 16(4): 709-18, 1995 May.
Artigo em Inglês | MEDLINE | ID: mdl-7476165

RESUMO

The thrS gene in Bacillus subtilis is specifically induced by starvation for threonine and is, in addition, autorepressed by the overproduction of its own gene product, the threonyl-tRNA synthetase. Both methods of regulation employ an antitermination mechanism at a factor-independent transcription terminator that occurs just upstream of the start codon. The effector of the induction mechanism is thought to be the uncharged tRNA(Thr), which has been proposed to base pair in two places with the leader mRNA to induce antitermination. Here we show that the autoregulation by synthetase overproduction is likely to utilize a mechanism similar to that characterized for induction by amino acid starvation, that is by altering the levels of tRNA charging in the cell. We also demonstrate that the base pairing interaction at the two proposed contact points between the tRNA and the leader are necessary but not always sufficient for either form of regulation. Finally, we present evidence that the thrS gene is expressed in direct proportion to the growth rate. This method of regulation is also at the level of antitermination but is independent of the interaction of the tRNA with the leader region.


Assuntos
Aminoacil-tRNA Sintetases/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Genes Bacterianos , Aminoacil-tRNA Sintetases/biossíntese , Bacillus subtilis/crescimento & desenvolvimento , Sequência de Bases , Códon/genética , Indução Enzimática , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Plasmídeos/genética , RNA Bacteriano/química , RNA Bacteriano/genética , RNA de Transferência/química , RNA de Transferência/genética
17.
Mol Microbiol ; 14(1): 31-40, 1994 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7830558

RESUMO

Expression of rpsO, the gene encoding the small ribosomal protein S15, is autoregulated at the translational level by S15, which binds to its mRNA in a region overlapping the ribosome-binding site. By measuring the effect of mutations on the expression of a translational rpsO-lacZ fusion and the S15 binding affinity for the translational operator, the formation of a pseudoknot in the operator site in vivo is fully demonstrated and appears to be a prerequisite for S15 binding. The mutational analysis suggests also that specific determinants for S15 binding are located in very limited regions of the structure formed by the pseudoknot. It is deduced that a specific pseudoknot conformation is a key element for autoregulation.


Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , Bacteriófago lambda/genética , Sequência de Bases , Sítios de Ligação , Códon , Análise Mutacional de DNA , Escherichia coli/metabolismo , Dados de Sequência Molecular , Plasmídeos , Ligação Proteica , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por Restrição , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/metabolismo , Transcrição Gênica , beta-Galactosidase/biossíntese
18.
J Biol Chem ; 269(10): 7473-82, 1994 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-7510287

RESUMO

The Bacillus subtilis cysE and cysS genes encoding, respectively, the serine acetyltransferase and the cysteinyl-tRNA synthetase were found downstream from the gltX gene encoding the glutamyl-tRNA synthetase. This gene organization is also conserved in Bacillus stearothermophilus where the cysE and cysS genes show high amino acid identity with those of B. subtilis. In both organisms the coding sequences of cysE and cysS overlap, suggesting a translational coupling. B. subtilis cysE and cysS were expressed in Escherichia coli using the inducible trc promoter; they functionally complement mutants of E. coli affected in those genes. Overproduction of B. subtilis CysRS in E. coli has a toxic effect on cell growth. Disruption of gltX and cysS by Campbell-type insertion is lethal for the cell, indicating that these genes code for an essential and unique function in B. subtilis. S1 mapping analysis shows that the transcription of gltX is under the control of a sigma A promoter located 43 base pairs upstream of the initiation codon. A T-box sequence and a rho-independent terminator known to regulate expression of other aminoacyl-tRNA synthetase genes and of some amino acid biosynthetic operons in Bacillus sp., were found between gltX and cysE. No sigma A promoter was detected upstream of cysE, which is consistent with the lethality of a Campbell-type insertion using a plasmid that interrupts transcription coming from the gltX promoter, and suggests that gltX, cysE, and cysS constitute an operon. This is the first case where genes implicated in the biosynthesis of an amino acid and its cognate aminoacyl-tRNA synthetase are shown to be co-transcribed.


Assuntos
Aminoacil-tRNA Sintetases/genética , Bacillus subtilis/genética , Cisteína/metabolismo , Glutamatos/metabolismo , Família Multigênica , Transcrição Gênica , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/metabolismo , Sequência de Bases , Clonagem Molecular , Cisteína/biossíntese , DNA Bacteriano , Escherichia coli/genética , Geobacillus stearothermophilus/genética , Ácido Glutâmico , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/química , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
19.
Mol Microbiol ; 11(2): 293-302, 1994 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8170391

RESUMO

During the IF2-catalysed formation of the 30S initiation complex, the GTP requirement and its subsequent hydrolysis during 70S complex formation are considered to be essential for translation initiation in Escherichia coli. In order to clarify the role of certain amino acid residues believed to be crucial for the GTP hydrolytic activity of E. coli IF2, we have introduced seven single amino acid substitutions into its GTP-binding site (Gly for Val-400; Thr for Pro-446; Gly, Glu, Gln for His-448; and Asn, Glu for Asp-501). These mutated IF2 proteins were expressed in vivo in physiological quantities and tested for their ability to maintain the growth of an E. coli strain from which the functional chromosomal copy of the infB gene has been deleted. Only one of the mutated proteins (Asp-501 to Glu) was able to sustain cell viability and several displayed a dominant negative effect. These results emphasize that the amino acid residues we substituted are essential for the IF2 functions and demonstrate the importance of GTP hydrolysis in translation initiation. These findings are discussed in relation to a previously proposed theoretical model for the IF2 G-domain.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Escherichia coli/genética , Genes Bacterianos , Genótipo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Fatores de Iniciação de Peptídeos/genética , Plasmídeos , Mutação Puntual , Fator de Iniciação 2 em Procariotos , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Mapeamento por Restrição
20.
J Bacteriol ; 176(2): 486-94, 1994 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8288542

RESUMO

The "housekeeping" threonyl-tRNA synthetase gene (thrS) of Bacillus subtilis is shown to be transcribed in vivo and in vitro from a single promoter. In vitro, 85% of all messages transcribed from the thrS promoter are terminated at a strong factor-independent terminator localized upstream of the thrS Shine-Dalgarno sequence, within the 305-nucleotide-long leader region. Overexpression of thrS represses transcriptional and translational thrS-lacZ fusions to a similar extent, suggesting that thrS is autoregulated at the transcriptional level. We show that autogenous control does not act at the level of transcription initiation but involves antitermination of the transcription mechanism. thrZ, the second threonyl-tRNA synthetase gene, is also autogenously regulated. However, the ability of the ThrS synthetase to repress thrS as well as thrZ expression is much greater than that of the ThrZ synthetase.


Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Isoenzimas/genética , Treonina-tRNA Ligase/genética , Bacillus subtilis/enzimologia , Sequência de Bases , Repressão Enzimática/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Dados de Sequência Molecular , Família Multigênica , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico/genética , Treonina-tRNA Ligase/biossíntese , Treonina-tRNA Ligase/farmacologia , Transcrição Gênica
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