title: Inhibition of Escherichia coli RNAp by T7 Gp2 protein: role of negatively charged strip of amino acid residues in Gp2.
abstract: Gp2, a 7 kDa protein encoded by T7 bacteriophage, is a potent inhibitor of Escherichia coli RNA polymerase (RNAp), the enzyme responsible for transcription of all bacterial genes and early viral genes. A prominent feature in the structure of Gp2 is a contiguous strip of seven negatively charged amino acid residues (negatively charged strip or NCS), located along one side of the molecule. The role of the NCS in Gp2 function is not known. Here, the in vivo and in vitro properties of altered forms of Gp2 with amino acid substitutions in the NCS are described. While mutations in the NCS do not compromise the folding or the ability of Gp2 to bind to the RNAp β' subunit, disruption of the NCS significantly attenuates Gp2 function in vivo and its ability to inhibit RNAp in vitro. Efficient inhibition of the RNAp by Gp2 also involves the amino terminal region 1 domain of the RNAp promoter specificity subunit σ(70), located in the vicinity of the primary Gp2 binding site in β'. The results are discussed in the context of hypothetical molecular mechanisms of RNAp inhibition by Gp2.
authors.affiliation: Section of Microbiology, Faculty of Medicine & Centre for Molecular Microbiology and Infection, Imperial College London, SW7 2AZ, UK.
authors.forename: Carol
authors.initials: C
authors.lastname: Sheppard
authors.forename: Beatriz
authors.initials: B
authors.lastname: Cámara
authors.forename: Andrey
authors.initials: A
authors.lastname: Shadrin
authors.forename: Natalia
authors.initials: N
authors.lastname: Akulenko
authors.forename: Minhao
authors.initials: M
authors.lastname: Liu
authors.forename: Geoff
authors.initials: G
authors.lastname: Baldwin
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
authors.forename: Ernesto
authors.initials: E
authors.lastname: Cota
authors.forename: Steve
authors.initials: S
authors.lastname: Matthews
authors.forename: Siva R
authors.initials: SR
authors.lastname: Wigneshweraraj
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D012808:Sigma Factor; C120334:gene 2 product, bacteriophage T7; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases