title: Visualizing the phage T4 activated transcription complex of DNA and E. coli RNA polymerase.
abstract: The ability of RNA polymerase (RNAP) to select the right promoter sequence at the right time is fundamental to the control of gene expression in all organisms. However, there is only one crystallized structure of a complete activator/RNAP/DNA complex. In a process called σ appropriation, bacteriophage T4 activates a class of phage promoters using an activator (MotA) and a co-activator (AsiA), which function through interactions with the σ(70) subunit of RNAP. We have developed a holistic, structure-based model for σ appropriation using multiple experimentally determined 3D structures (Escherichia coli RNAP, the Thermus aquaticus RNAP/DNA complex, AsiA /σ(70) Region 4, the N-terminal domain of MotA [MotA(NTD)], and the C-terminal domain of MotA [MotA(CTD)]), molecular modeling, and extensive biochemical observations indicating the position of the proteins relative to each other and to the DNA. Our results visualize how AsiA/MotA redirects σ, and therefore RNAP activity, to T4 promoter DNA, and demonstrate at a molecular level how the tactful interaction of transcriptional factors with even small segments of RNAP can alter promoter specificity. Furthermore, our model provides a rational basis for understanding how a mutation within the β subunit of RNAP (G1249D), which is far removed from AsiA or MotA, impairs σ appropriation.
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA Department of Biochemistry and Molecular Pharmacology, NYU Langone Medical Center, New York University School of Medicine, 180 Varick Street, Room 637, New York, NY 10014, USA.
authors.forename: Tamara D
authors.initials: TD
authors.lastname: James
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, NYU Langone Medical Center, New York University School of Medicine, 180 Varick Street, Room 637, New York, NY 10014, USA timothy.cardozo@med.nyu.edu.
authors.forename: Timothy
authors.initials: T
authors.lastname: Cardozo
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Abell
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Meng-Lun
authors.initials: ML
authors.lastname: Hsieh
authors.affiliation: Collaborative Protein Technology Resource, Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Lisa M Miller
authors.initials: LM
authors.lastname: Jenkins
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Saheli S
authors.initials: SS
authors.lastname: Jha
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA dhinton@helix.nih.gov.
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Hinton
mesh_terms: D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000378:metabolism*; D055592:Biophysical Phenomena; D003432:Cross-Linking Reagents / Q000378:metabolism; D004247:DNA / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008027:Light; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
chemical_list: D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D010455:Peptides; D004247:DNA; D012321:DNA-Directed RNA Polymerases