A hybrid sigma subunit directs RNA polymerase to a hybrid promoter in Escherichia coli.
Most of the sigma (transcriptional initiation specificity) subunits of RNA polymerase, from a wide range of eubacteria, show strong elements of amino acid sequence similarity. There is evidence that two of the "conserved" regions, 2.4 and 4.2, are involved in recognition of the consensus DNA sequences centred near -10 and -35, respectively, which define promoter sites for the initiation of transcription. Since all the alternative sigma subunits of the above type function by binding to a common core polymerase enzyme in a given bacterium, it can be predicted that a hybrid sigma might be functional, and if so should permit RNA polymerase to initiate only at a correspondingly hybrid promoter. To test these predictions, a hybrid gene encoding the amino-proximal 529 amino acids of the major Escherichia coli sigma protein, sigma 70 (including region 2.4) followed by the last 82 amino acids of the heat-shock sigma protein, sigma 32 (including region 4.2) was constructed and fused to Plac on a plasmid. Major-consensus, heat-shock and hybrid promoters were fused to a chloramphenicol acetyl transferase (CAT) reporter gene on a compatible plasmid. CAT assays showed that, as predicted, a promoter with a "heat-shock" -35 consensus and a "major" -10 consensus sequence (PHM) required Plac-dependent production of the hybrid sigma (sigma 70-32) for activity in vivo. PHM then became a strong promoter. The hybrid sigma gene has potential advantages over its parents for structure-function studies.
Kumar
A
A
Institute of Cell and Molecular Biology, University of Edinburgh, Scotland, U.K.
Grimes
B
B
Logan
M
M
Wedgwood
S
S
Williamson
H
H
Hayward
R S
RS
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000502:physiology*; D001483:Base Sequence; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000502:physiology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012808:Sigma Factor / Q000502:physiology*; D014157:Transcription Factors
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C069774:heat-shock sigma factor 32; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases