## 3 _E. coli_\(\sigma\)[70] Promoter Recognition
### Bending the Downstream Duplex into the Cleft  
In RPc, the downstream duplex (\(-5\) to \(+20\)) is unprotected from OH and DNase, indicating that it is not interacting with the RNAP cleft [58, 123, 124, 125, 126, 127]. By contrast, at \(\lambda\)Pr, the OH footprint of an advanced closed complex (like I\({}_{1,\mathrm{L}}\) in Figure 3, obtained in real time [27]) is periodic and extends from \(-80\) to \(+20\), indicating that one face of the downstream duplex is in the cleft or otherwise protected by RNAP. Closed complexes with similar downstream footprint boundaries (between \(+15\) and \(+25\)) are observed at other promoters and have been variously called RPi (at _rrnB_ P1 [130, 131], _lac_UV5 [127], and T7A1 [126]), RPc (at _\(\lambda\)prmup-1_\(\Delta 265\)[132]) and RPc (at the \(\sigma^{32}\) promoter _groE_[58, 125]). In the conversion of RPc to I\({}_{1,\mathrm{L}}\) (Figure 3) the downstream DNA must be bent by at least 90\({}^{\circ}\) at the upstream end of the \(-10\) element to enter the cleft [16, 60]. What bends the duplex is not known. The \(-11\)A base flips out of the stacked duplex and into a pocket of \(\sigma^{70}\) region 2 [9, 10, 17]. If base flipping occurs before this region is bent, it would introduce a point of greater flexibility into the duplex, resulting in spontaneous bending of the duplex and its capture by the cleft. Alternatively, bending of the \(-10\) region by RNAP may occur first, destabilizing the stacking and resulting in base flipping. If the interaction of \(\sigma^{70}\) region 2 with the unbent \(-10\) region in early closed complexes were limited to the \(-12\) position, this interaction might either induce flipping of the adjacent \(-11\)A base or dictate the site of bending and subsequent base flipping after a downstream interaction [10, 97]. In-cleft elements like \(\sigma_{1.1}\) and downstream mobile elements (DME; see below) of free RNAP holoenzyme are positioned (Figure 2) to prevent entry of nonpromoter DNA into the active site cleft. These elements of RNAP appear to be part of an extensive, sophisticated network of upstream and downstream interactions that regulate the access of the downstream duplex to the cleft to form I\({}_{1,\mathrm{L}}\). Conversion of RPc to I\({}_{1,\mathrm{L}}\) appears to involve a series of closed intermediates with different extents of downstream interaction, in which the extent of bending into the cleft may be coupled to the extent of upstream bending and wrapping discussed above. This regulatory network responds to promoter sequence and structure, as well as factor binding and solution conditions. Many of these same elements may also be involved in the regulation of open complex lifetime (see below). Several lines of evidence exist for this network. As discussed above, truncation of upstream DNA greatly reduces the rate of the isomerization step of open complex formation. In addition to this, truncation of \(\lambda\)Pr at \(-47\) shortens the downstream DNase footprint boundary of the advanced closed complex ensemble from \(+20\) to \(+7\) on the nontemplate strand and \(+2\) on the template strand, indicating that only the upstream half of the downstream duplex (\(-10\) to \(+2\)/\(+7\)) is inserted in the cleft in this closed complex [75]. Similarly, a RNAP variant lacking \(\sigma_{1.1}\) forms a closed complex footprint at early times at \(\lambda\)P\({}_{\rm R}\), with a downstream boundary of \(\sim-5\)[47]. Thus, upstream DNA interactions and \(\sigma_{1.1}\) facilitate bending of the downstream duplex into the cleft. Similarly, at _rmB_ P1, the complex formed at equilibrium in the absence of nucleotides at 12 \({}^{\circ}\)C has a downstream boundary which is approximately \(+1\)[130, 131]. This footprint boundary requires that there be some bending at the \(-10\) element, but not enough to fully load bases \(-10\) to \(+20\) into the cleft. What could be the reason for this partial bending in these complexes?