## 3 _E. coli_\(\sigma\)[70] Promoter Recognition  
_In vivo_, initiation rates vary at least 10,000-fold for different promoters [61, 62]. Rates of open complex formation (at a specified [RNAP]) and dissociation _in vitro_ both span a similar range determined by the sequence and structure of the promoter DNA [61, 62]. For a given promoter sequence, changes in temperature, salt, and solute concentrations [63, 64, 24, 65, 66], as well as additions of protein factors and ligands, can affect these kinetics by 10-1000-fold or more. Promoter elements have been defined structurally, genetically, and/or functionally (summarized in Figure 1). What promoter regions are most important for which steps in the mechanism? As an extension of the bipartite proposal of promoter function [67], a working hypothesis is that promoter sequence and structure upstream of (and including at least part of) the \(-10\) element direct the steps of initial binding of RNAP and subsequent conformational changes that culminate in bending of the downstream duplex into the cleft. These steps precede the central DNA opening step, which opens approximately 13 bp (\(-11\) to \(+2\) at \(\lambda\)P\({}_{\textrm{R}}\)) of the promoter DNA. Collectively these steps determine the rate of open complex formation. For some (but not all) promoters, promoter elements in and downstream of the \(-10\) element direct post-opening steps of the mechanism (in particular open complex stabilization) that determine the lifetime of the open complex. All of these steps are discussed in subsequent sections. The farthest upstream sequence-specific interactions between RNAP and promoter DNA are in the UP element region from approximately base \(-40\) to \(-60\) (Figure 1, cyan; reviewed in [68]). UP elements are phased AT-tracts recognized by the C-terminal domains of the \(\alpha\) subunits (\(\alpha\)CTDs); the narrow minor groove at these sequences favors specific \(\alpha\)CTD binding [69]. UP elements were first noted for their ability to increase transcription from the rRNA promoters, and a consensus UP element sequence determined using mutational analysis increased transcription 330-fold _in vivo_[70, 71]. The entire UP element consists of two subsites, proximal and distal, one for each \(\alpha\)CTD; promoters may have one or both, although the distal UP element tends to function nearly as well as a full UP element [72]. Because of the curvature generally observed at phased A- or T-tracts, it was hypothesized that the main function of UP elements was to bend the DNA. However, not all UP elements display significant curvature [73] as even single base disruptions in an A- or T-tract can practically abolish bending [74]. At \(\lambda\)P\({}_{\textrm{R}}\) and T7A1, real time and low temperature DNase and hydroxyl radical (OH) footprinting of closed complexes reveals that recognition of the \(-35\), \(-10\), and UP elements induces bending of upstream DNA at \(-38\) and \(-48\)[27, 59, 75] and wrapping of the region from \(\sim\)\(-60\) to \(-80\) on RNAP [25, 26, 27, 59, 75, 76]. Structural evidence for bending at the \(-35\) element is provided by [43, 77], and a discussion of functional bending by the \(\alpha\)CTDs is provided in [68]. These upstream interactions are very important for efficient open complex formation at \(\lambda\)P\({}_{\textrm{R}}\) and _lac_UV5 (see below) [75, 78]. The \(-35\) element (Figure 1, blue) has the consensus sequence 5'-TTGACA-3' [79] with the \(-35\)T, \(-34\)T, and \(-33\)G being the most highly conserved [80]. These bases interact via the major groove with a helix-turn-helix motif of \(\mathfrak{s}_{4.2}\) (Figures 1 and 2, blue), which bends the DNA approximately \(36^{\circ}\) in cocrystal structures of the \(-35\) element and _Thermus aquatica_\(\mathfrak{s}_{4.2}\)[13, 43] and a recent initiation complex structure with _E. coli_ holoenzyme [77]. Strong DNase I footprints enhancements suggestive of bending are also observed at the upstream end of the \(-35\) element at \(\lambda\)P\({}_{\textrm{R}}\)[75].