Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

• Published in: Proceedings of the National Academy of Sciences - PNAS 2009-11, Vol.106 (47), p.19830-19835
• Main Authors: Hudson, Brian P, Quispe, Joel, Lara-González, Samuel, Kim, Younggyu, Berman, Helen M, Arnold, Eddy, Ebright, Richard H, Lawson, Catherine L
• Format: Article
• Language: English
• Subjects: Base Sequence; BASIC BIOLOGICAL SCIENCES; Biological Sciences; Coordinate systems; Crystal structure; Cyclic AMP receptor protein; Cyclic AMP Receptor Protein - chemistry; Cyclic AMP Receptor Protein - ultrastructure; Deoxyribonucleic acid; Dimers; DNA; DNA, Bacterial - chemistry; DNA, Bacterial - ultrastructure; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - ultrastructure; E coli; ELECTRON MICROSCOPY; Enzymes; ESCHERICHIA COLI; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - ultrastructure; GENE REGULATION; GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; Macromolecular Substances - chemistry; Models, Molecular; Molecular Sequence Data; national synchrotron light source; Nucleic Acid Conformation; Promoter regions; Promoter Regions, Genetic; PROMOTERS; Protein Structure, Tertiary; Protein subunits; Protein Subunits - chemistry; PROTEINS; Ribonucleic acid; RNA; RNA polymerase; RNA POLYMERASES; STAINS; TRANSCRIPTION; Transcription, Genetic; Transcriptional activation
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0908782106

We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions -78 to +20 of a Class I CAP-dependent promoter with a CAP site at position -61.5 and a premelted transcription bubble. A 20-Å electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP α subunit C-terminal domain (αCTD), interactions of αCTD with σ⁷⁰ region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP β', β, and σ⁷⁰ subunits.