Construction and functional analyses of a comprehensive σ54 site-directed mutant library using alanine–cysteine mutagenesis

Abstract

The σ54 factor associates with core RNA polymerase (RNAP) to form a holoenzyme that is unable to initiate transcription unless acted on by an activator protein. σ54 is closely involved in many steps of activator-dependent transcription, such as core RNAP binding, promoter recognition, activator interaction and open complex formation. To systematically define σ54 residues that contribute to each of these functions and to generate a resource for site specific protein labeling, a complete mutant library of σ54 was constructed by alanine–cysteine scanning mutagenesis. Amino acid residues from 3 to 476 of Cys(-)σ54 were systematically mutated to alanine and cysteine in groups of two adjacent residues at a time. The influences of each substitution pair upon the functions of σ54 were analyzed in vivo and in vitro and the functions of many residues were revealed for the first time. Increased σ54 isomerization activity seldom corresponded with an increased transcription activity of the holoenzyme, suggesting the steps after σ54 isomerization, likely to be changes in core RNAP structure, are also strictly regulated or rate limiting to open complex formation. A linkage between core RNAP-binding activity and activator responsiveness indicates that the σ54-core RNAP interface changes upon activation.