title: Substitutions in the Escherichia coli RNA polymerase inhibitor T7 Gp2 that allow inhibition of transcription when the primary interaction interface between Gp2 and RNA polymerase becomes compromised.
abstract: The Escherichia coli-infecting bacteriophage T7 encodes a 7 kDa protein, called Gp2, which is a potent inhibitor of the host RNA polymerase (RNAp). Gp2 is essential for T7 phage development. The interaction site for Gp2 on the E. coli RNAp is the β' jaw domain, which is part of the DNA binding channel. The binding of Gp2 to the β' jaw antagonizes several steps associated with interactions between the RNAp and promoter DNA, leading to inhibition of transcription at the open promoter complex formation step. In the structure of the complex formed between Gp2 and a fragment of the β' jaw, amino acid residues in the β3 strand of Gp2 contribute to the primary interaction interface with the β' jaw. The 7009 E. coli strain is resistant to T7 because it carries a charge reversal point mutation in the β' jaw that prevents Gp2 binding. However, a T7 phage encoding a mutant form of Gp2, called Gp2(β), which carries triple amino acid substitutions E24K, F27Y and R56C, can productively infect this strain. By studying the molecular basis of inhibition of RNAp from the 7009 strain by Gp2(β), we provide several lines of evidence that the E24K and F27Y substitutions facilitate an interaction with RNAp when the primary interaction interface with the β' jaw is compromised. The proposed additional interaction interface between RNAp and Gp2 may contribute to the multipronged mechanism of transcription inhibition by Gp2.
authors.affiliation: Section of Microbiology and MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, UK.
authors.forename: Andrey
authors.initials: A
authors.lastname: Shadrin
authors.affiliation: Section of Microbiology and MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, UK.
authors.forename: Carol
authors.initials: C
authors.lastname: Sheppard
authors.affiliation: Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia.|Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.|Waksman Institute for Microbiology and Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
authors.affiliation: Section of Microbiology and MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, UK.
authors.forename: Steve
authors.initials: S
authors.lastname: Matthews
authors.affiliation: Section of Microbiology and MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, UK.
authors.forename: Sivaramesh
authors.initials: S
authors.lastname: Wigneshweraraj
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017123:Bacteriophage T7 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D014158:Transcription, Genetic; D014764:Viral Proteins
chemical_list: D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; C120334:gene 2 product, bacteriophage T7; D012321:DNA-Directed RNA Polymerases