## 3 _E. coli_\(\sigma\)[70] Promoter Recognition
### Open Complex Stabilization  
The conversion of the relatively unstable initial open complex (I\({}_{2}\)) to the highly stable open complex (RP\({}_{\rm O}\)) at \(\lambda\)P\({}_{\rm R}\) involves conformational changes in DNA and RNAP. Comparison of permanganate footprints of \(\lambda\)P\({}_{\rm R}\) I\({}_{2}\) and RP\({}_{\rm O}\) indicates that the discriminator region of the nontemplate strand is repositioned in the cleft, becoming more reactive in RP\({}_{\rm O}\)[23, 95]. The large dependences of the equilibrium constant K\({}_{3}\) for this step on urea and salt concentration indicate the folding/assembly of approximately 120 RNAP residues of the jaw and other DME on the downstream duplex [134]. Comparative quantitative studies with \(\lambda\)P\({}_{\rm R}\) truncated at base \(+12\) and a \(\beta\)' jaw deletion mutant of RNAP indicate that the \(\beta\)' jaw DME [127](Figure 2, brown) and \(\beta\)' sequence insertion 3 DME (also termed \(\beta\)' insertion 6; Figure 2, green) are involved in the assembly-DNA binding process that converts I\({}_{2}\) to the much more stable RPo at \(\lambda\)Pr. In this model, supported by studies using RNAP and DNA heteroduplexes of varying lengths [29, 30, 31, 135], the DME assemble on downstream DNA, stabilizing the open complex. Recent evidence suggests that the interaction of DNA between positions +4 and +6 with switch regions 1 and 2 may trigger cleft closure and DME folding/assembly and clamping [29]. Kinetic dissection of open complex stabilization at \(\lambda\)Pr led to the discovery of a third on-pathway intermediate designated I\({}_{3}\)[24]. Formation of I\({}_{3}\) from I\({}_{2}\) involves the majority (approximately two-thirds) of the folding and assembly of RNAP DME involved in converting I\({}_{2}\) to RPo. The extent of this assembly is intriguingly similar to that observed for the open complex formed by a \(\beta\)' jaw deletion variant, which may therefore be an analog of I\({}_{3}\)[134]. Another potential analog, based on similarity in lifetime and downstream interactions, might be the open complex formed at T7A1 [18, 136]. The lifetime of the open complex formed with \(rmB\) P1 (\(\sim\)1 s) [95] is intriguingly similar to that of I\({}_{2}\)[24], consistent with the idea that the \(rmB\) P1 sequence (in particular the weak discriminator and short spacer) precludes the stabilizing interaction from cleft closing and DME assembly induced by promoters like \(\lambda\)Pr and T7A1. Further experiments are required to explore the roles of promoter elements in the mechanism, and the potential relevance of I\({}_{2}\), I\({}_{3}\) and other forms of the open complex to transcription initiation and its control. Figure 4: Mechanism of transcription initiation: resolvable kinetic steps. Minimal mechanisms of open complex formation and dissociation, showing by color coding the steps that contribute to the observed rate constants (k\({}_{\rm obs}\), k\({}_{\rm d}\)) for promoters like \(\lambda\)Pr that form a stable open complex RP\({}_{\rm O}\). For the common experimental situation of excess RNAP, the rate constant for open complex formation (k\({}_{\rm obs}\)) is determined by the front half of the mechanism (boxed in purple), including the equilibrium constant K\({}_{1}\) for formation of the ensemble of closed (I\({}_{1}\)) intermediates, the forward rate constant k\({}_{2}\) of the isomerization step that includes DNA opening, and the excess RNAP concentration. Likewise, k\({}_{\rm d}\) is determined by the late steps of the mechanism (boxed in green), including the rate constant k\({}_{\rm 2}\) for DNA closing and the equilibrium constant K\({}_{3}\) for stabilization of the initial open complex I\({}_{2}\) to form longer-lived I\({}_{3}\) and/or RP\({}_{\rm O}\) complexes [65, 66]. Forming and Stabilizing the Open Complex; Regulation of the Kinetics by Promoter Sequence, Length and Other Variables  
Kinetic studies are essential to any determination of mechanism, together with structural information about intermediates.