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

摘要

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 lgG-binding domain of Staphyfococcus 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-hy- drated helical crystals. Comparison of the mutant RNAP structure with the previously determined wild-type RNAP structure by Fou- rier 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.