title: A basic/hydrophobic cleft of the T4 activator MotA interacts with the C-terminus of E.coli sigma70 to activate middle gene transcription.
abstract: Transcriptional activation often employs a direct interaction between an activator and RNA polymerase. For activation of its middle genes, bacteriophage T4 appropriates Escherichia coli RNA polymerase through the action of two phage-encoded proteins, MotA and AsiA. Alone, AsiA inhibits transcription from a large class of host promoters by structurally remodelling region 4 of sigma(70), the primary specificity subunit of E. coli RNA polymerase. MotA interacts both with sigma(70) region 4 and with a DNA element present in T4 middle promoters. AsiA-induced remodelling is proposed to make the far C-terminus of sigma(70) region 4 accessible for MotA binding. Here, NMR chemical shift analysis indicates that MotA uses a 'basic/hydrophobic' cleft to interact with the C-terminus of AsiA-remodelled sigma(70), but MotA does not interact with AsiA itself. Mutations within this cleft, at residues K3, K28 and Q76, both impair the interaction of MotA with sigma(70) region 4 and MotA-dependent activation. Furthermore, mutations at these residues greatly decrease phage viability. Most previously described activators that target sigma(70) directly use acidic residues to engage a basic surface of region 4. Our work supports accumulated evidence indicating that 'sigma appropriation' by MotA and AsiA uses a fundamentally different mechanism to activate transcription.
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Molecular and Cellular Biology, NIDDK, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Richard P
authors.initials: RP
authors.lastname: Bonocora
authors.forename: Gregori
authors.initials: G
authors.lastname: Caignan
authors.forename: Christopher
authors.initials: C
authors.lastname: Woodrell
authors.forename: Milton H
authors.initials: MH
authors.lastname: Werner
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Hinton
mesh_terms: D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D017122:Bacteriophage T4 / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000821:virology*; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D009682:Magnetic Resonance Spectroscopy; D050296:Microbial Viability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
chemical_list: C078876:AsiA protein, Enterobacteria phage T4; D004268:DNA-Binding Proteins; C067996:MotA protein, Enterobacteria phage T4; D012808:Sigma Factor; D014157:Transcription Factors; D014764:Viral Proteins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase