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

Abstract

Recent determinations of the structures of the bacterial RNA polymerase (RNAP) and promoter complex thereof establish that RNAP functions as a complex molecular machine that contains distinct structural modules that undergo major conformational changes during transcription. However, the contribution of the RNAP structural modules to transcription remains poorly understood. The bacterial core RNAP (␣ 2 ␤␤; E) associates with a sigma () subunit to form the holoenzyme (E Multisubunit DNA-dependent RNA polymerases (RNAP) 1 are complex molecular machines that synthesize a RNA copy from a DNA template. In Escherichia coli, five subunits (␣ 2 ␤␤Ј) form the RNAP catalytic core (E) that associates with a sigma () subunit to form the holoenzyme (E). The subunit imparts on the core RNAP the ability to specifically recognize and initiate transcription from promoters. Biochemical and structural studies indicate that the protein-protein contacts at the interface between core RNAP and , an extensive and functionally specialized sets of surfaces, govern the conformational changes that allow efficient promoter recognition and transcription initiation (1-3).Sequence comparisons reveal two unrelated families of RNAP factors. Members of the major family of factors form RNAP holoenzymes that recognize promoters and form transcriptionally competent promoter complexes in the absence of other factors or energy sources. This family, which includes most bacterial factors is named after the prototypical housekeeping of E. coli, 70 . 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). The importance of these conformational changes is underlined by our recent observation that removal of the E. coli RNAP ␤ flap domain abolished the ability of the mutant E 70 to recognize promoters of the Ϫ10/Ϫ35 class (13). E 54 recognizes and binds promoters containing conserved consensus elements centered around Ϫ24 and Ϫ12 nucleotides upstream of the transcription start site at ϩ1. These promoter elements can be considered as functional analogues of the Ϫ35/Ϫ10 consensus promoter elements recognized by E 70 cla...

Mapping σ54-RNA Polymerase Interactions at the –24 Consensus Promoter Element

“…However, both 54 -and 54 -RNAP bind equally well to the early melted promoter probe (12,37,40). We previously demonstrated that the RNAP ␤-subunit flap domain contributes (either directly or indirectly) to the creation of the DNA fork junction at the Ϫ12 promoter region (31). We propose that during closed complex formation, 54 Region I and promoter DNA undergo core RNAP-directed conformational changes involving the ␤-subunit flap domain that result in (i) creation of the DNA fork junction at the Ϫ12 promoter region and (ii) the tight binding of the 54 Region I to the DNA fork junction.…”

“…E. coli core RNAP containing the ␤-subunit flap deletion (⌬885-914) was purified as described in Ref. 31. Wild-type E. coli core RNAP was purchased from Epicentre Technologies.…”

“…To investigate the contribution of the ␤-subunit flap domain to promoter interactions by 54 , we reconstituted 54 Cys 463 -RNAP and Cys 46 -RNAP using a mutant RNAP core enzyme deleted for the flap domain (21,31 (Fig. 8a, lanes 2, 4, and 6).…”

Nucleotide-dependent Triggering of RNA Polymerase-DNA Interactions by an AAA Regulator of Transcription

“…6c). Binding of 54 or 54 -RNAP to the S. meliloti nifH homoduplex probe or probe 1 (Table Ia) response to ATP hydrolysis by the activator (11,14,19).…”

“…OP-Cu is a minor groove-specific DNA cleaving reagent that has been widely used to study 54 -RNAP interactions with the Ϫ12 promoter region (6,19). In closed promoter complexes formed with the 54 -RNAP on homoduplex promoter probe (Table Ia), the minor groove at the Ϫ12 position is distorted and thus is susceptible to cleavage by OP-Cu (6).…”