RESUMO
Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Aminoácidos de Cadeia Ramificada/química , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Cristalografia por Raios X , Pegada de DNA , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Isoleucina/química , Isoleucina/metabolismo , Estrutura Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Estrutura Secundária de ProteínaRESUMO
Transcription antitermination in the ribosomal operons of Escherichia coli results in the modification of RNA polymerase by specific proteins, altering its basic properties. For such alterations to occur, signal sequences in rrn operons are required as well as individual interacting proteins. In this study we tested putative rrn transcription antitermination-inducing sequences from five different bacteria for their abilities to function in E. coli. We further examined their response to the lack of one known rrn transcription antitermination protein from E. coli, NusB. We monitored antitermination activity by assessing the ability of RNA polymerase to read through a factor-dependent terminator. We found that, in general, the closer the regulatory sequence matched that of E. coli, the more likely there was to be a successful antitermination-proficient modification of the transcription complex. The rrn leader sequences from Pseudomonas aeruginosa, Bacillus subtilis, and Caulobacter crescentus all provided various levels of, but functionally significant antitermination properties to, RNA polymerase, while those of Mycobacterium tuberculosis and Thermotoga maritima did not. Possible RNA folding structures of presumed antitermination sequences and specific critical bases are discussed in light of our results. An unexpected finding was that when using the Caulobacter crescentus rrn leader sequence, there was little effect on terminator readthrough in the absence of NusB. All other hybrid antitermination system activities required this factor. Possible reasons for this finding are discussed.
Assuntos
Evolução Molecular , Óperon/genética , RNA Ribossômico/genética , Regiões Terminadoras Genéticas/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/genética , Especificidade da Espécie , Transcrição GênicaRESUMO
CodY, a global regulator of gene expression in low G + C Gram-positive bacteria, was found to repress toxin gene expression in Clostridium difficile. Inactivation of the codY gene resulted in derepression of all five genes of the C. difficile pathogenicity locus during exponential growth and stationary phase. CodY was found to bind with high affinity to a DNA fragment containing the promoter region of the tcdR gene, which encodes a sigma factor that permits RNA polymerase to recognize promoters of the two major toxin genes as well as its own promoter. CodY also bound, but with low affinity, to the toxin gene promoters, suggesting that the regulation of toxin gene expression by CodY occurs primarily through direct control of tcdR gene expression. Binding of CodY to the tcdR promoter region was enhanced in the presence of GTP and branched-chain amino acids, suggesting a link between nutrient limitation and the expression of C. difficile toxin genes.