Structure of the RNA polymerase assembly factor Crl and identification of its interaction surface with sigma S

Abstract

Bacteria utilize multiple sigma factors that associate with core RNA polymerase (RNAP) to control transcription in response to changes in environmental conditions. In Escherichia coli and Salmonella enterica, Crl positively regulates the σS regulon by binding to σS to promote its association with core RNAP. We recently characterized the determinants in σS responsible for specific binding to Crl. However, little is known about the determinants in Crl required for this interaction. Here, we present the X-ray crystal structure of a Crl homolog from Proteus mirabilis in conjunction with in vivo and in vitro approaches that probe the Crl-σS interaction in E. coli. We show that the P. mirabilis, Vibrio harveyi, and E. coli Crl homologs function similarly in E. coli, indicating that Crl structure and function are likely conserved throughout gammaproteobacteria. We utilize phylogenetic conservation and bacterial two-hybrid analyses to predict residues in Crl important for the interaction with σS. The results of p-benzoylphenylalanine (BPA)-mediated UV cross-linking studies further support the model in which an evolutionarily conserved central cleft is the surface on Crl that binds to σS. Within this conserved binding surface, we identify a key residue in Crl that is critical for activation of EσS-dependent transcription in vivo and in vitro. Our study provides a physical basis for understanding the σS-Crl interaction. © 2014, American Society for Microbiology.