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Journal of Bacteriology, October 2005, p. 6762-6769, Vol. 187, No. 19
0021-9193/05/$08.00+0     doi:10.1128/JB.187.19.6762-6769.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Mutational Analysis of Escherichia coli Heat Shock Transcription Factor Sigma 32 Reveals Similarities with Sigma 70 in Recognition of the –35 Promoter Element and Differences in Promoter DNA Melting and –10 Recognition

Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, Ohio 44106-4935

Received 24 May 2005/ Accepted 20 July 2005

Upon the exposure of Escherichia coli to high temperature (heat shock), cellular levels of the transcription factor ³² rise greatly, resulting in the increased formation of the ³² holoenzyme, which is capable of transcription initiation at heat shock promoters. Higher levels of heat shock proteins render the cell better able to cope with the effects of higher temperatures. To conduct structure-function studies on ³² in vivo, we have carried out site-directed mutagenesis and employed a previously developed system involving ³² expression from one plasmid and a ß-galactosidase reporter gene driven by the ³²-dependent groE promoter on another in order to monitor the effects of single amino acid substitutions on ³² activity. It was found that the recognition of the –35 region involves similar amino acid residues in regions 4.2 of E. coli ³² and ⁷⁰. Three conserved amino acids in region 2.3 of ³² were found to be only marginally important in determining activity in vivo. Differences between ³² and ⁷⁰ in the effects of mutation in region 2.4 on the activities of the two sigma factors are consistent with the pronounced differences between both the amino acid sequences in this region and the recognized promoter DNA sequences.

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