Coupling nucleotide hydrolysis to transcription activation performance in a bacterial enhancer binding protein

Abstract

The bacterial enhancer binding proteins (bEBP) are members of the AAA+ protein family and have a highly conserved 'DE' Walker B motif thought to be involved in the catalytic function of the protein with an active role in nucleotide hydrolysis. Based on detailed structural data, we analysed the functionality of the conserved 'DE' Walker B motif of a bEBP model, phage shock protein F (PspF), to investigate the role of these residues in the σ54-dependent transcription activation process. We established their role in the regulation of PspF self-association and in the relay of the ATPase activity to the remodelling of an RNA polymerase·promoter complex (Eσ54·DNA). Specific substitutions of the conserved glutamate (E) allowed the identification of new functional ATP· bEBP·Eσ54 complexes which are stable and transcriptionally competent, providing a new tool to study the initial events of the σ54-dependent transcription activation process. In addition, we show the importance of this glutamate residue in σ54·DNA conformation sensing, permitting the identification of new intermediate stages within the transcription activation pathway. © 2007 The Authors.