## 3 _E. coli_\(\sigma\)[70] Promoter Recognition
Flipping of base \(-11\)A into its pocket may thus nucleate bending of the DNA into the cleft before opening at some promoters; alternatively, other RNAP-DNA interactions may first bend the DNA, nucleating \(-11\)A flipping [10]. A recent comparative kinetic study of all 4096 \(-10\) variants [22] finds that RNAP interactions with \(-7\)T play a relatively stronger role in open complex formation kinetics, suggesting a model in which bubble nucleation occurs at \(-7\)T, or \(-7\)T flipping stabilizes the bubble in the transition state, or both. While \(-11\)A and \(-7\)T are the most conserved bases of the \(-10\) element [79, 88, 101], they are not absolutely required for relatively fast initiation kinetics, particularly if the \(-10\) element is sufficiently AT-rich [22]. A recent structure shows that _E. coli_\(\sigma^{\rm E}\) uses a similar base flipping mechanism to recognize, and possibly nucleate opening at, the \(-10\) element [102]. The discriminator region between the \(-10\) element and the start site (Figure 1, orange) is involved in regulation of open complex lifetime. Its upstream end interacts with \(\sigma_{1.2}\)[9, 17, 77, 95, 103] (Figures 1 and 2, orange). Most discriminators are 6-8 bases in length [88]. Mutational analyses [104, 105, 106] indicate that lifetime decreases as discriminator length increases from 6 to 8 bases. A bioinformatic analysis of _E. coli_ promoters [107] found that the frequency of discriminators also decreases with increasing length from 6 to 8 bases. These observations together indicate that many _E. coli_ promoters form long-lived open complexes. More recent efforts to comprehensively map the transcription start sites in _E. coli_[108] will no doubt lead to elucidation of the _in vivo_ importance of the sequence of this region. Structural data indicate that for a six-base discriminator, \(-6\)G on the nontemplate strand flips into a pocket on the surface of \(\sigma_{1.2}\)[17]. The \(-5\) nontemplate strand base is also very important for open complex stability, with a G being relatively stabilizing and a C being relatively destabilizing [95]. Similarly, \(\sigma^{\rm A}\) from _T. aquaticus_ binds most favorably to a \(5\)'-GGG-\(3\)' sequence immediately downstream of the \(-10\) element [109]. The rRNA promoters generally have a C on the nontemplate strand two bases downstream of the \(-10\) element [110], leading to short open complex lifetimes; this sequence element is important for their regulation _in vitro_ and _in vivo_[95]. A recent crystal structure of a model open complex identifies a \"core recognition element\" (or \"CRE\", bases \(-4\) to \(+2\) on the nontemplate strand) recognized by the \(\beta\) subunit [17]. Base \(+2\)G is flipped into a pocket, increasing the lifetime of an RNAP-fork junction complex containing a consensus \(-10\) element and strong discriminator [17]. Each CRE base except \(-1\) appears to be specifically recognized, and substitutions in the corresponding \(\beta\) residues lead to defects in transcription initiation. No consensus sequence of CRE has been detected [87] and thymine bases in this region are generally observed to be reactive to permanganate, implying that they are exposed to solvent. However, _in vivo_ footprints argues that CRE interacts with RNAP during elongation [111] and base-specific interactions involving the CRE have been implicated in pausing [112]. The most common transcription start site base is A \(\geq\) G \(>\) T \(>>\) C [106]. Subsaturating concentrations of the initiating nucleotides affect the kinetics of initiation, just as the concentration of any substrate affects the velocity V of an enzyme-catalyzed reaction for subsaturating concentrations (where V \(<\) V\({}^{\rm max}\)). In addition, binding of initiating NTP stabilizes the short-lived open complex at rRNA promoters [113], shifting the distribution of promoter complexes from closed to open in a NTP concentration-dependent manner.