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.
Upon the exposure of Escherichia coli to high temperature (heat shock), cellular levels of the transcription factor sigma32 rise greatly, resulting in the increased formation of the sigma32 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 sigma32 in vivo, we have carried out site-directed mutagenesis and employed a previously developed system involving sigma32 expression from one plasmid and a beta-galactosidase reporter gene driven by the sigma32-dependent groE promoter on another in order to monitor the effects of single amino acid substitutions on sigma32 activity. It was found that the recognition of the -35 region involves similar amino acid residues in regions 4.2 of E. coli sigma32 and sigma70. Three conserved amino acids in region 2.3 of sigma32 were found to be only marginally important in determining activity in vivo. Differences between sigma32 and sigma70 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.
Kourennaia
Olga V
OV
Center for RNA Molecular Biology, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4973, USA.
Tsujikawa
Laura
L
Dehaseth
Pieter L
PL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics; D018833:Chaperonins; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000502:physiology; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic / Q000502:physiology*; D017433:Protein Structure, Secondary; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D012808:Sigma Factor; C069774:heat-shock sigma factor 32; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D018833:Chaperonins