title: Characterization of the interactions between the bacteriophage T4 AsiA protein and RNA polymerase.
abstract: The anti-sigma factor AsiA effects a change in promoter specificity of the Escherichia coli RNA polymerase via interactions with two conserved regions of the sigma(70) subunit, denoted 4.1 and 4.2. Free AsiA is a symmetrical homodimer. Here, we show that AsiA is monomeric when bound to sigma(70) and that a subset of the residues that contribute to the homodimer interface also contributes to the interface with sigma(70). AsiA interacts primarily with C-terminal sections of regions 4.1 and 4.2, which show remarkable sequence similarity. An AsiA monomer can simultaneously, and apparently cooperatively, bind both isolated regions 4.1 and 4.2 at preferred, distinct subsites, whereas region 4.1 alone or region 4.2 alone can interact with either subsite. These results suggest structural and functional plasticity in the interaction of AsiA with sigma(70) and support the notion of discrete roles for regions 4.1 and 4.2 in transcription regulation by AsiA. Furthermore, we show that AsiA inhibits recognition of the -35 consensus promoter element by region 4 of sigma(70) indirectly, as the residues on region 4 responsible for AsiA binding are distinct from those involved in DNA binding. Finally, we show that AsiA must directly disrupt the interaction of region 4 with the RNA polymerase beta subunit flap domain, resulting in a distance change between region 2 and region 4 of sigma(70). Thus, a new paradigm for transcription regulation by AsiA is emerging, whereby the distance between the DNA binding domains in sigma(70) is regulated, and promoter recognition specificity is modulated, by mediating the interactions of the sigma region 4 with the beta subunit flap domain.
authors.affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
authors.forename: Mario F
authors.initials: MF
authors.lastname: Simeonov
authors.forename: Ramona J
authors.initials: RJ
authors.lastname: Bieber Urbauer
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Gilmore
authors.forename: Karen
authors.initials: K
authors.lastname: Adelman
authors.forename: Edward N
authors.initials: EN
authors.lastname: Brody
authors.forename: Anita
authors.initials: A
authors.lastname: Niedziela-Majka
authors.forename: Leonid
authors.initials: L
authors.lastname: Minakhin
authors.forename: Tomasz
authors.initials: T
authors.lastname: Heyduk
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Urbauer
mesh_terms: D017122:Bacteriophage T4 / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000821:virology; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: C078876:AsiA protein, Enterobacteria phage T4; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases