title: The 5.5 protein of phage T7 inhibits H-NS through interactions with the central oligomerization domain.
abstract: The 5.5 protein (T7p32) of coliphage T7 (5.5(T7)) was shown to bind and inhibit gene silencing by the nucleoid-associated protein H-NS, but the mechanism by which it acts was not understood. The 5.5(T7) protein is insoluble when expressed in Escherichia coli, but we find that 5.5(T7) can be isolated in a soluble form when coexpressed with a truncated version of H-NS followed by subsequent disruption of the complex during anion-exchange chromatography. Association studies reveal that 5.5(T7) binds a region of H-NS (residues 60 to 80) recently found to contain a distinct domain necessary for higher-order H-NS oligomerization. Accordingly, we find that purified 5.5(T7) can disrupt higher-order H-NS-DNA complexes in vitro but does not abolish DNA binding by H-NS per se. Homologues of the 5.5(T7) protein are found exclusively among members of the Autographivirinae that infect enteric bacteria, and despite fairly low sequence conservation, the H-NS binding properties of these proteins are largely conserved. Unexpectedly, we find that the 5.5(T7) protein copurifies with heterogeneous low-molecular-weight RNA, likely tRNA, through several chromatography steps and that this interaction does not require the DNA binding domain of H-NS. The 5.5 proteins utilize a previously undescribed mechanism of H-NS antagonism that further highlights the critical importance that higher-order oligomerization plays in H-NS-mediated gene repression.
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 1A8 Canada.
authors.forename: Sabrina S
authors.initials: SS
authors.lastname: Ali
authors.forename: Emily
authors.initials: E
authors.lastname: Beckett
authors.forename: Sandy Jeehoon
authors.initials: SJ
authors.lastname: Bae
authors.forename: William Wiley
authors.initials: WW
authors.lastname: Navarre
mesh_terms: D017123:Bacteriophage T7 / Q000502:physiology*; D004926:Escherichia coli / Q000502:physiology* / Q000821:virology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D036781:Fimbriae Proteins / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C529458:FimG protein, E coli; D014764:Viral Proteins; C080078:gene 5.5 protein, Bacteriophage T7; D036781:Fimbriae Proteins