Isolation and characterization of mutations in region 1.2 of Escherichia coli σ70

Abstract

Eubacterial RNA polymerase uses the sigma (σ) subunit for recognition of and transcription initiation from promoter DNA sequences. One family of s factors includes those related to the primary s factor from Escherichia coli, σ70. Members of the σ70 family have four highly conserved domains, of which regions 2 to 4 are present in all members. Region 1 can be subdivided into regions 1.1 and 1.2. Region 1.1 affects DNA binding by σ70 alone, as well as transcription initiation by holoenzyme. Region 1.2, present and highly conserved in most sigma factors, has not yet been assigned a putative function, although previous work has demonstrated that it is not required for either association with the core subunits of RNA polymerase or promoter-specific binding by holoenzyme. We generated random single amino acid substitutions targeted to region 1.2 of E. coli s70 as well as a deletion of region 1.2, and characterized the behaviour of the mutant s factors both in vivo and in vitro to investigate the function of region 1.2 during transcription initiation. In this study, we show that mutations in region 1.2 can affect promoter binding, open complex and initiated complex formation and the transition from abortive transcription to elongation.