Direct localization of a β-subunit domain on the three-dimensional structure of Escherichia coli RNA polymerase

^*Laboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10021; and ^†Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison, WI 53706

Communicated by Jeffrey W. Roberts, Cornell University, Ithaca, NY (received for review August 9, 1999)

Abstract

To identify the location of a domain of the β-subunit of Escherichia coli RNA polymerase (RNAP) on the three-dimensional structure, we developed a method to tag a nonessential surface of the multisubunit enzyme with a protein density easily detectable by electron microscopy and image processing. Four repeats of the IgG-binding domain of Staphylococcus aureus protein A were inserted at position 998 of the E. coli RNAP β-subunit. The mutant RNAP supported E. coli growth and showed no apparent functional defects in vitro. The structure of the mutant RNAP was determined by cryoelectron microscopy and image processing of frozen-hydrated helical crystals. Comparison of the mutant RNAP structure with the previously determined wild-type RNAP structure by Fourier difference analysis at 20-Å resolution directly revealed the location of the inserted protein domain, thereby locating the region around position 998 of the β-subunit within the RNAP three-dimensional structure and refining a model for the subunit locations within the enzyme.

Footnotes

↵ ‡ Present address: Waksman Institute and Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ 08854.
↵ § To whom reprint requests should be addressed. E-mail: darst{at}rockvax.rockefeller.edu.
Abbreviations:

DR,

dispensable region;

EM,

electron microscopy;

Rifr/s,

rifampicin resistant/sensitive;

RNAP,

RNA polymerase;

SPA,

Staphylococcus aureus protein A