In vitro transcription from the Escherichia coli ilvIH promoter.

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RESUMO

Lrp (leucine-responsive regulatory protein) activates the expression of the Escherichia coli ilvIH operon in vivo and mediates the repression of the operon by exogenous leucine. In previous studies, operon expression in vivo was measured with transcriptional fusions of lacZ to the ilvIH promoter. Here, ilvIH mRNA was measured directly by primer extension. The steady-state level of ilvIH mRNA was 11-fold higher in a wild-type parent strain than in a derivative lacking Lrp. A two-step procedure was developed for measuring ilvIH mRNA synthesized in vitro. RNA was synthesized with plasmid templates and purified RNA polymerase, and then ilvIH mRNA was measured by primer extension. In vitro, mRNA synthesis was initiated at two sites, one corresponding to the in vivo site (promoter P1) and the other corresponding to a site about 60 bp further upstream (promoter P2). Purified Lrp stimulated transcription two- to fivefold from promoter P1, whereas it decreased transcription more than fivefold from promoter P2. Transcription from promoter P1 was stimulated by Lrp with templates containing the wild-type ilvIH promoter but not with templates containing mutations in an Lrp binding site. Furthermore, under at least some conditions, leucine reversed the stimulatory effect of Lrp. Taken together with the results of mutational analyses, these results establish that Lrp acts directly to stimulate transcription from the ilvIH promoter. Furthermore, they suggest that the ilvIH promoter is recognized by a sigma 70 RNA polymerase.

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