11] σ Factors: Purification and DNA binding

Abstract

Publisher Summary This chapter focuses on purification of σ polypeptides using affinity tags, and analysis of DNA binding in vitro . The sigma (σ) subunit of prokaryotic RNA polymerases provides specificity for transcription initiation by directing polymerase to the appropriate promoter DNA sequences. The chapter describes a large number of different σ subunits, which are related to the Escherichia coli primary σ. Members of this group are characterized by four regions of amino acid similarity. The ability of σ to bind to DNA in vitro , in the absence of the core polymerase subunits varies depending on the structure of the σ polypeptide. For example, DNA binding by σ is not observed unless the amino-terminal inhibitory domain is removed. Alternative σ factors, such as Bacillus subtilis σ K and Salmonella typhimurium σ, which lack the inhibitory domain, bind to DNA in full-length form. Another family of σ factors, related to E. coli σ, contains members that are quite dissimilar to the σ family in both structure and function. Unlike σ, full-length σ is capable of binding to DNA.