The X‐ray Crystal Structure of Escherichia Coli RNA Polymerase σ70 Holoenzyme

Abstract

Escherichia coli RNA polymerase (RNAP) is the best‐studied bacterial RNAP and has been used as the model RNAP for potential RNAP‐targeting antibiotic screening and evaluation. However, the X‐ray crystal structure of E. coli RNAP has been limited to individual domains. In this study, E. coli RNAP core enzyme (E) and holoenzyme (Eσ70) were prepared from overexpressed proteins. The holoenzyme was crystallized and solved its X‐ray structure at 3.8 Å resolution. The E. coli holoenzyme structure shows the σ region 1.1 (σ1.1) and α subunit C‐terminal domain (αCTD) structures for first time in any bacterial RNAP. The structural comparison between the E. coli and Thermus RNAPs shows that the ω subunit structure is different in E. coli from that in Thermus that may be related to the fact that the only E. coli RNAP is able to respond ppGpp for transcription regulation. The σ1.1 is positioned at the RNAP DNA binding channel and completely blocks DNA entry to the RNAP active site. The structure reveals that the σ1.1 contains a basic patch on its surface, which may play an important role in the DNA interaction to facilitate the transition from the closed to open promoter complexes. The αCTD is positioned next to the σ domain 4 (σ4) with a fully stretched linker between the N‐ and C‐terminal domains. E. coli RNAP crystals can be conveniently prepared from overexpressed proteins, allowing further structural studies of bacterial RNAP mutants, including antibiotic resistant RNAPs.