C OMMUNICATION Inhibition of Escherichia coli RNAp by T7 Gp2 Protein: Role of Negatively Charged Strip of Amino Acid Residues in Gp2 Carol Sheppard 1 †, Beatriz Cámara 1 †, Andrey Shadrin 2,3,4 , Natalia Akulenko 3,4 , Minhao Liu 5 , Geoff Baldwin 6 , Konstantin Severinov 3,4 , Ernesto Cota 5 , Steve Matthews 5 and Siva R Wigneshweraraj 1 ⁎ 1 Section of Microbiology, Faculty of Medicine & Centre for Molecular Microbiology and Infection, Imperial College London, SW7 2AZ, UK 2 Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Science Pushchino, Russia 3 Waksman Institute for Microbiology and Department of Molecular Biology and Biochemistry, Rutgers, State University of New Jersey, Piscataway, NJ 08854, USA 4 Institutes of Molecular Genetics and Gene Biology, Russian Academy of Sciences, Moscow, Russia 5 Division of Molecular Biosciences, Imperial College London, SW7 2AZ, UK 6 Centre for Structural Biology, Imperial College London, SW7 2AZ, UK Received 19 November 2010; received in revised form 3 February 2011; accepted 4 February 2011 Available online 18 February 2011 Edited by: R. Ebright Keywords: bacteriophage T7; RNA polymerase; RNA polymerase inhibitor; T7 Gp2; transcription regulation 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. © 2011 Elsevier Ltd. All rights reserved. Transcription of DNA is catalysed by the DNA- dependent RNA polymerase (RNAp). Bacterial RNAp is a multisubunit enzyme comprised of a five-subunit catalytic core (α 2 ββ′ω; abbreviated E) and one of several σ subunits, each conferring upon the catalytic core an ability to initiate transcription from specific DNA sites called promoters. 1 Most bacterial promoters with conserved sequences near *Corresponding author. E-mail address: s.r.wig@imperial.ac.uk. † Contributed equally to this work. Abbreviations used: RNAp, RNA polymerase; RPc, closed promoter complex; RPo, open promoter complex; NCS, negatively charged strip; DBC, DNA-binding channel; FP, fluorescence polarization. doi:10.1016/j.jmb.2011.02.013 J. Mol. Biol. (2011) 407, 623–632 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb 0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.