RESUMO
The response regulator Spo0A is the master control element in the initiation of sporulation in Bacillus subtilis. Like many other multi-domain response regulators, the latent activity of the effector, C-terminal domain is stimulated by phosphorylation on a conserved aspartic acid residue in the regulatory, N-terminal domain. If a threshold concentration of phosphorylated Spo0A is achieved, the transcription of genes required for sporulation is activated, whereas the genes encoding stationary phase sentinels are repressed, and sporulation proceeds. Despite detailed genetic, biochemical and structural characterisation, it is not understood how the phosphorylation signal in the receiver domain is transduced into DNA binding and transcription activation in the distal effector domain. An obstacle to our understanding of Spo0A function is the uncertainty concerning changes in quaternary structure that accompany phosphorylation. Here we have revisited this question and shown unequivocally that Spo0A forms dimers upon phosphorylation and that the subunit interactions in the dimer are mediated principally by the receiver domain. Purified dimers of two mutants of Spo0A, in which the phosphorylatable aspartic acid residue has been substituted, activate transcription from the spoIIG promoter in vitro, whereas monomers do not. This suggests that dimers represent the activated form of Spo0A.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator sigma , Esporos Bacterianos/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Bacillus subtilis/enzimologia , Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Cromatografia em Gel , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Dimerização , Genes Bacterianos/genética , Genes Reguladores/genética , Modelos Biológicos , Modelos Moleculares , Peso Molecular , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Fatores de Transcrição/genética , UltracentrifugaçãoRESUMO
The unusual architecture of the enzyme (MsAcT) isolated from Mycobacterium smegmatis forms the mechanistic basis for favoring alcoholysis over hydrolysis in water. Unlike hydrolases that perform alcoholysis only under anhydrous conditions, MsAcT demonstrates alcoholysis in substantially aqueous media and, in the presence of hydrogen peroxide, has a perhydrolysis:hydrolysis ratio 50-fold greater than that of the best lipase tested. The crystal structures of the apoenzyme and an inhibitor-bound form have been determined to 1.5 A resolution. MsAcT is an octamer in the asymmetric unit and forms a tightly associated aggregate in solution. Relative to other structurally similar monomers, MsAcT contains several insertions that contribute to the oligomerization and greatly restrict the shape of the active site, thereby limiting its accessibility. These properties create an environment by which MsAcT can catalyze transesterification reactions in an aqueous medium and suggests how a serine hydrolase can be engineered to be an efficient acyltransferase.
Assuntos
Aciltransferases/química , Álcoois/química , Mycobacterium smegmatis/enzimologia , Água/química , Acetatos/química , Aciltransferases/genética , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Cristalografia por Raios X , Peróxido de Hidrogênio/química , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium smegmatis/genética , Fenilcarbamatos/química , Propilenoglicóis/química , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Triglicerídeos/químicaRESUMO
A simple and highly efficient method was developed to produce a library of Escherichia coli clones that express a particular chromosomal gene at a wide range of expression levels. The basic strategy was to replace all or part of the upstream region of a coding sequence containing the elements involved in its expression (promoter, operator, gene coding for a regulator, ribosome binding site, and start codon) with a PCR-generated library of expression cassettes.