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

doi:10.1073/pnas.0908782106

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

^aDepartment of Chemistry and Chemical Biology and
^bCenter for Advanced Biotechnology and Medicine, Rutgers University, 610 Taylor Road, Piscataway, NJ 08854 ;
^cNational Resource for Automated Molecular Microscopy, The Scripps Research Institute, CB 129, 10550 North Torrey Pines Rd, La Jolla, CA 92037; and
^dWaksman Institute and
^eHoward Hughes Medical Institute, Rutgers University, Piscataway, NJ 08854

Edited by Seth A. Darst, Rockefeller University, New York, NY, and approved October 05, 2009 (received for review August 3, 2009)

Abstract

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.

Footnotes

¹To whom correspondence should be addressed. E-mail: cathy.lawson{at}rutgers.edu
Author contributions: B.P.H., H.M.B., E.A., R.H.E., and C.L.L. designed research; B.P.H., J.Q., and C.L.L. performed research; S.L., Y.K., and R.H.E. contributed new reagents/analytic tools; B.P.H. and C.L.L. analyzed data; and B.P.H., R.H.E., and C.L.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The EM reconstruction volume has been deposited in the Electron Microscopy Data Bank, www.emdatabank.org (EMD ID code EMD-5127); the EM fitted coordinate model has been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3iyd).
This article contains supporting information online at www.pnas.org/cgi/content/full/0908782106/DCSupplemental.