Characterization of the Escherichia coli transcription factor sigma 70: localization of a region involved in the interaction with core RNA polymerase

Abstract

A set of internal deletions and frame-shift mutations was made in the structural gene for the major sigma factor of Escherichia coli RNA polymerase (sigma 70). The truncated proteins from these various mutants were examined to determine if they retained the ability to bind core RNA polymerase. Two assays were used to determine core-binding activity. Gel filtration was used to separate free sigma 70 from sigma 70 bound to core polymerase. Immunoprecipitation of polymerase using an anti-alpha-subunit monoclonal antibody was also used to determine if the various truncated proteins were bound to core. Results from these experiments indicate core-binding activity is retained when large portions of the sigma 70 protein are deleted. Deletion of a region in the central portion of the protein caused a large decrease in core-binding activity. The results suggest that the region spanning amino acids 361-390 is important for efficient core-binding activity. Sequence comparison of various sigma factors shows highly conserved amino acids in this region. A synthetic peptide having the sequence of amino acids 361-390 was synthesized and examined for the ability to bind core RNA polymerase.