RESUMO
The high activity of the rrnB P1 promoter in Escherichia coli results from a cis-acting DNA sequence, the UP element, and a trans-acting transcription factor, FIS. In this study, we examine the effects of FIS and the UP element at the other six rrn P1 promoters. We find that UP elements are present at all of the rrn P1 promoters, but they make different relative contributions to promoter activity. Similarly, FIS binds upstream of, and activates, all seven rrn P1 promoters but to different extents. The total number of FIS binding sites, as well as their positions relative to the transcription start site, differ at each rrn P1 promoter. Surprisingly, the FIS sites upstream of site I play a much larger role in transcription from most rrn P1 promoters compared to rrnB P1. Our studies indicate that the overall activities of the seven rrn P1 promoters are similar, and the same contributors are responsible for these high activities, but these inputs make different relative contributions and may act through slightly different mechanisms at each promoter. These studies have implications for the control of gene expression of unlinked multigene families.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Escherichia coli , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , RNA Ribossômico/biossíntese , Óperon de RNAr , Sequência de Bases , Sítios de Ligação , Fator Proteico para Inversão de Estimulação , Fatores Hospedeiros de Integração , Dados de Sequência Molecular , RNA Bacteriano/biossíntese , Elementos de Resposta , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/fisiologia , Ativação TranscricionalAssuntos
Bactérias/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , RNA Ribossômico/genética , Transcrição Gênica , Sequência de Bases , Sequência Consenso , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Bacteriano/genética , Fatores de Transcrição/metabolismoRESUMO
A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.