title: Direct localization of a beta-subunit domain on the three-dimensional structure of Escherichia coli RNA polymerase.
abstract: To identify the location of a domain of the beta-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 beta-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-A resolution directly revealed the location of the inserted protein domain, thereby locating the region around position 998 of the beta-subunit within the RNAP three-dimensional structure and refining a model for the subunit locations within the enzyme.
authors.affiliation: Laboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
authors.forename: N
authors.initials: N
authors.lastname: Opalka
authors.forename: R A
authors.initials: RA
authors.lastname: Mooney
authors.forename: C
authors.initials: C
authors.lastname: Richter
authors.forename: K
authors.initials: K
authors.lastname: Severinov
authors.forename: R
authors.initials: R
authors.lastname: Landick
authors.forename: S A
authors.initials: SA
authors.lastname: Darst
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D004925:Escherichia / Q000737:chemistry / Q000201:enzymology*; D008854:Microscopy, Electron; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D012321:DNA-Directed RNA Polymerases