title: 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 σ(S) regulon by binding to σ(S) to promote its association with core RNAP. We recently characterized the determinants in σ(S) 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-σ(S) 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 σ(S). 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 σ(S). Within this conserved binding surface, we identify a key residue in Crl that is critical for activation of Eσ(S)-dependent transcription in vivo and in vitro. Our study provides a physical basis for understanding the σ(S)-Crl interaction.
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Amy B
authors.initials: AB
authors.lastname: Banta
authors.affiliation: Biosciences Division, Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne, Illinois, USA.
authors.forename: Marianne E
authors.initials: ME
authors.lastname: Cuff
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Hueylie
authors.initials: H
authors.lastname: Lin
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Angela R
authors.initials: AR
authors.lastname: Myers
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Wilma
authors.initials: W
authors.lastname: Ross
authors.affiliation: Biosciences Division, Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne, Illinois, USA.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Joachimiak
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA rgourse@bact.wisc.edu.
authors.forename: Richard L
authors.identifier: http://orcid.org/0000-0002-9675-5314
authors.initials: RL
authors.lastname: Gourse
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005075:Biological Evolution; D017124:Conserved Sequence; D003460:Crystallization; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011513:Proteus mirabilis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; C077481:Crl protein, Bacteria