title: Conserved regions 4.1 and 4.2 of sigma(70) constitute the recognition sites for the anti-sigma factor AsiA, and AsiA is a dimer free in solution.
abstract: The association of the bacteriophage T4-encoded AsiA protein with the final sigma(70) subunit of the Escherichia coli RNA polymerase is one of the principal events governing transcription of the T4 genome. Analytical ultracentrifugation and NMR studies indicate that free AsiA is a symmetric dimer and the dimers can exchange subunits. Using NMR, the mutual recognition sites on AsiA and final sigma(70) have been elucidated. Residues throughout the N-terminal half of AsiA are involved either directly or indirectly in binding to final sigma(70) whereas the two highly conserved C-terminal regions of final sigma(70), denoted 4.1 and 4.2, constitute the entire AsiA binding domain. Peptides corresponding to these regions bind tightly to AsiA individually and simultaneously. Simultaneous binding promotes structural changes in AsiA that mimic interaction with the complete AsiA binding determinant of final sigma(70). Moreover, the results suggest that a significant rearrangement of the dimer accompanies peptide binding. Thus, both conserved regions 4.1 and 4.2 are intimately involved in recognition of AsiA by final sigma(70). The interaction of AsiA with 4.1 provides a potential explanation of the differential abilities of DNA and AsiA to bind to free final sigma(70) and a mechanistic alternative to models of AsiA function that rely on binding to a single site on final sigma(70).
authors.affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA. jurbauer@ku.edu
authors.forename: J L
authors.initials: JL
authors.lastname: Urbauer
authors.forename: K
authors.initials: K
authors.lastname: Adelman
authors.forename: R J
authors.initials: RJ
authors.lastname: Urbauer
authors.forename: M F
authors.initials: MF
authors.lastname: Simeonov
authors.forename: J M
authors.initials: JM
authors.lastname: Gilmore
authors.forename: M
authors.initials: M
authors.lastname: Zolkiewski
authors.forename: E N
authors.initials: EN
authors.lastname: Brody
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D012808:Sigma Factor / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: C078876:AsiA protein, Enterobacteria phage T4; D012808:Sigma Factor; D012996:Solutions; D014764:Viral Proteins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases