Conformational flexibility of bacterial RNA polymerase

Abstract

The structure of Escherichia coli core RNA polymerase (RNAP) was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 Å. Because of the high sequence conservation between the core RNAP subunits, we were able to interpret the E. coli structure in relation to the highresolution x-ray structure of Thermus aquaticus core RNAP. A very large conformational change of the T. aquaticus RNAP x-ray structure, corresponding to opening of the main DNA͞RNA channel by nearly 25 Å, was required to fit the E. coli map. This finding reveals, at least partially, the range of conformational flexibility of the RNAP, which is likely to have functional implications for the initiation of transcription, where the DNA template must be loaded into the channel.

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

“…We propose that DksA-D74 functions during initiation by neutralizing the positive charges of β-R678/ R1106 and altering the dense network of polar-electrostatic interactions in the immediate vicinity of the active center (23,26,28,29). This could alter the conformation of two neighboring mobile elements of β, fork loop-1 and fork loop-2, destabilizing the intermediate on the pathway to open complex formation.…”

Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

“…S2 A and B). T. thermophilus RNAP and E. coli RNAP share Ϸ40% sequence identity and have roughly the same shape and size, but also have well-characterized species-specific differences (27)(28)(29)(30)(31) (Fig. S2C).…”

“…S2C). Trial 3D reconstructions were judged in part by the appearance of expected E. coli species-specific domains-in particular, ␤ dispensable region 1 (␤DR1; also known as SI1) (27)(28)(29), ␤ dispensable region 2 (␤DR2; also known as SI2) (27)(28)(29)31), ␤Ј trigger-loop nonconserved domain (␤ЈGNCD; also known as SI3) (28)(29)(30), and the 70 nonconserved-region insert within 70 region 2 ( 70 NCR) (32). The final reconstruction was created from 13,841 particles in 280 classes (Fig.…”

“…For ␤DR2, a volume consistent with the expected mass is obtained only at lower contour levels, suggesting partial disorder. By comparison, in an EM structure of E. coli RNAP core (27), ␤DR1 is partially disordered, ␤DR2 is ordered, and ␤ 1126-1179 appears to be disordered.…”

Multiple Roles of the RNA Polymerase β Subunit Flap Domain in ς54-Dependent Transcription

“…Members of the second, minor family of factors, the 54 family, form RNAP holoenzymes that recognize promoters but require additional protein factors and a source of energy in the form of ATP or GTP hydrolysis for formation of transcriptionally competent promoter complexes (4 -6). Despite the differences in pathways that lead to transcription-competent open promoter complexes, both classes of factors occupy similar positions within their respective RNAP holoenzymes and appear to utilize some common RNAP surfaces for transcription initiation (7-10).Binding of a 70 family factor induces conformational changes within the core RNAP (2,11,12). Structural modules of the core RNAP, designated as the ␤Ј clamp, the ␤ flap, and the ␤ lobes, interact with a 70 family subunit ( A ) in the structures of Thermus aquaticus and Thermus thermophilus RNAP holoenzymes and undergo conformational changes, which orientate and position 70 DNA-binding domains within the RNAP holoenzyme to allow promoter recognition (2, 3).…”