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

To identify the location of a domain of the beta-subunit of Escherichia coi l RNA polymerase (RNAP) on the three-dimensional structure, we developed a method to tag a nonessential surface of the multisubunit enzyme with a prot ein density easily detectable by electron microscopy and image processing. Four repeats of the IgG-binding domain of Staphylococcus aureus protein A w ere inserted at position 998 of the E. coil RNAP beta-subunit, The mutant R NAP supported E. coil growth and showed no apparent functional defects in v itro. The structure of the mutant RNAP was determined by cryoelectron micro scopy 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-Angstrom 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-dimen sional structure and refining a model for the subunit locations within the enzyme.