title: Architecture of the bacteriophage T4 activator MotA/promoter DNA interaction during sigma appropriation.
abstract: Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called σ appropriation, the T4 co-activator AsiA structurally remodels the σ(70) subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ(70) and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the σ-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotA(NTD), MotA(CTD), and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual "double wing" motif present within MotA(CTD) resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses.
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Meng-Lun
authors.initials: ML
authors.lastname: Hsieh
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892; Structural Biology Program, Sackler Institute, New York University Langone Medical Center, New York, New York 10016.
authors.forename: Tamara D
authors.initials: TD
authors.lastname: James
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Leslie
authors.initials: L
authors.lastname: Knipling
authors.affiliation: Hartwell Center for Bioinformatics and Biotechnology.
authors.forename: M Brett
authors.initials: MB
authors.lastname: Waddell
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
authors.forename: Stephen
authors.initials: S
authors.lastname: White
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892. Electronic address: dhinton@helix.nih.gov.
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Hinton
mesh_terms: D020816:Amino Acid Motifs; D017122:Bacteriophage T4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000821:virology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli 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
keywords: AsiA; Bacteriophage; DNA-binding Protein; FeBABE; MotA; RNA Polymerase; Sigma; Transcription; Transcription Regulation
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