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/genéticaRESUMO
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 RestriçãoRESUMO
The T4 mot gene regulates middle mode RNA synthesis in phage-infected cells. The mot gene product has been identified in two ways. (i) Infections with amber and temperature-sensitive mot mutants both lead to the disappearance of a number of protein bands on SDS-polyacrylamide gels. These are middle mode proteins whose synthesis depends on mot function. The mot protein disappears from such gels after infection with a mot amber mutant, but not with the mot missense mutant. (ii) This same protein is the only one to have a charge alteration when proteins from wild-type phage and mot missense mutant infections are compared by two-dimensional gel electrophoresis. Mot protein is basic and has a mol. wt. of 24 000. It migrates between the positions of gp 1 and gp IPIII on 15% SDS-polyacrylamide gels. Mot protein synthesis begins immediately after infection and continues until 4 min after infection at 30 degrees C, after which time it is strongly inhibited. This inhibition depends neither on T4 DNA synthesis nor on ADP ribosylation of the alpha subunits of the Escherichia coli RNA polymerase. The mot protein does not regulate its own biosynthesis. It is stable throughout the course of infection.
