## 2 Bacterial RNAP \(\sigma^{70}\) Holoenzyme  
_E. coli_ RNAP core enzyme is a five-subunit 370 kDa assembly (\(\alpha\beta\beta\omega\)) [36]; for an extensive review of this structure, see [37]. RNAP is shaped roughly like a crab claw, with an active site cleft running between the \(\beta\)' and \(\beta\) subunits [38]. Although the active site and the overall crab claw shape of RNAPs are highly conserved across all kingdoms [39, 40], much of the enzyme is not. The surface of the RNAP is highly divergent, and in many organisms large insertions are present in the \(\beta\), \(\beta\)' and \(\sigma\) subunits [41]. The functions of many of these regions have yet to be identified. RNAP core enzyme carries out all steps of transcription except promoter-specific initiation, which requires an accessory \(\sigma\) factor. There are seven \(\sigma\) factors in _E. coli_, which mediate transcription of distinct sets of genes (reviewed in [5, 42]). Holoenzyme containing \(\sigma^{70}\) (termed the \"housekeeping \(\sigma\)\") transcribes most of the genes required for growth and cell maintenance, whereas others have more specialized roles, e.g., during stress and nutrient limitation. By itself \(\sigma^{70}\) is incapable of promoter binding and DNA opening. Binding to RNAP core enzyme unmasks specificity determinants in \(\sigma^{70}\)[14, 16] and creates the functional holoenzyme. As part of holoenzyme, \(\sigma^{70}\) recognizes a promoter composed of two well-conserved sequence elements, the \(-10\) and \(-35\) hexamers (Figure 1), which are separated by a 16-19 bp spacer region. \(\sigma^{70}\) is folded into four domains connected by flexible linkers [43]. Upon binding of \(\sigma\) to core, the domains remain independently folded while the linkers extend [13, 14, 16]. Each domain of \(\sigma^{70}\) interacts with both RNAP and DNA; domains 2 and 4 make specific contacts with the \(-10\) and \(-35\) regions, respectively [43], while domain 3 interacts with the extended \(-10\) region present in some promoters [44]. The flexible N-terminal \(\sigma^{70}\) region 1.1 (\(\sigma_{1.1}\); Figure 2, purple) consists of a three-helix bundle connected by a flexible linker to \(\sigma_{1.2}\)[45]. \(\sigma_{1.1}\), found only in the homologous group 1 \(\sigma\) factors, autoinhibits free \(\sigma\) factor from binding to DNA in the absence of core [46] and occupies the active site cleft in the absence of DNA [8, 12]. Kinetics experiments with RNAP lacking \(\sigma_{1.1}\) indicate that this mobile element increases both the isomerization rate (including DNA opening) [47] and the dissociation rate of open complexes [18, 19]. In crystal structures [8, 12, 14, 16], the active site cleft is \(\sim\)70 A deep and 100 A long, but only \(\sim\)15 A wide. Early cryo-EM structures [48] and a recent FRET study in solution [49] reveal that the cleft is dynamic, with a significant fraction of holoenzyme molecules displaying a much wider (20-25 A) cleft. This flexibility is likely due to the movement of comparatively rigid RNAP \"modules\" relative to each other. Multisubunit RNAPs contain five \"switch\" regions [39, 40], which undergo conformational changesduring open complex formation, altering relative positions of the modules. Wider clefts, resulting largely from different positioning of the \"clamp\" module, have been observed in a paused complex [50] and a complex with transcription factor Gfh1 [51]. Opening of the clamp has been proposed to underlie the mechanism of action by the stress alarmone ppGpp [52, 53]. Several antibiotics inhibit modular movements of RNAP by binding to or near these switch regions [54, 55, 56]. Opening of the RNAP clamp likely permits loading of the duplex DNA during initiation. Since the hydrated DNA duplex is about 25 A wide, early models of open complex formation proposed that DNA opening occurred outside the cleft before entry of the strands [13, 37, 57].