## 1 Introduction  
Transcription is the first step of gene expression and is therefore one of the most fundamental processes of life. All living organisms, as well as many viruses, encode at least one RNA polymerase (RNAP) enzyme that synthesizes an RNA copy of the template DNA; the core structure and many mechanistic and regulatory elements are shared by bacterial, eukaryotic and archaeal RNAPs (reviewed in [1,2]). Bacterial RNAPs are composed of a core enzyme, which carries out RNA synthesis, and a specificity (\(\sigma\)) subunit for recognition of promoter DNA sequence and subsequent events of initiation (Figures 1 and 2). Specific binding to promoter DNA forms an initial closed complex and sets in motion a series of large conformational changes which bend the downstream duplex DNA into the active site cleft of RNAP and then open the DNA to form a transcription bubble, placing the template DNA strand into the active site. At some (but not all) promoters, subsequent conformational changes in RNAP and the nontemplate strand stabilize this initial open complex (reviewed in [3]). Many steps of this process are highly regulated by promoter DNA sequence, accessory protein factors and small ligands, nucleotide concentration, temperature, salt and solute concentrations, and other environmental variables; aspects of regulation are reviewed in [1,4,5,6,7]. Figure 1: Sequence-specific interactions between \(\sigma^{70}\) RNAP and regions of the promoter. Schematic representations of the subunits of RNAP core, \(\sigma^{70}\), and promoter DNA. RNAP: \(\alpha\): cyan; \(\beta\) and \(\beta\): gray; \(\omega\): black. \(\sigma\) regions: as shown. Promoter: UP element: cyan; \(-35\) element: blue; extended \(-10\): red; \(-10\) element: yellow; discriminator: orange; transcription start site: green; DNA downstream of the transcription start site: gray. Linker regions in \(\alpha\) and \(\sigma\) subunits are shown as springs. Nontemplate strand sequences of a “consensus” and \(\lambda\)P\({}_{\mathrm{R}}\), T7A1 and \(rrn\)B P1 promoters are shown below; missing bases are indicated by dashes. A detailed understanding of the mechanism of initiation and its regulation is centrally important throughout biology, with many applications in biotechnology, pharmacology, and medicine. Determination of noncovalent mechanisms like that of open complex formation and stabilization require both kinetic and structural studies. High resolution structural studies provide detailed information about reactants and products, as well as any intermediates that can be trapped and stabilized [8, 9, 10, 11, 12, 13, 14, 15, 16, 17], but intermediates in open complex formation have been challenging to obtain and characterize in this way. Kinetic studies are used to investigate effects of promoter and RNAP variants, regulators, and regulatory variables [18, 19, 20, 21, 22], as well as to establish reaction conditions necessary to obtain transient high concentrations (bursts) of key intermediates for characterization by footprinting and other methods [23, 24]. Real-time DNA footprinting provides snapshots of the population of intermediates (and any reactants and products) at any given time, as well as kinetic information about the change in the distribution of these species with time [23, 25, 26, 27]. Ensemble and single-molecule experiments with fluorescent probes can provide unparalleled combinations of structural, dynamic and kinetic-mechanistic information [21, 28, 29, 30, 31, 32, 33, 34, 35]. Studies with _Escherichia coli_ RNAP reveal that the early steps of open complex formation, including initial specific binding to the promoter and some or all of the coupled conformational changes that bend DNA into the cleft, are often rapidly reversible in comparison to the slower \"isomerization\" step that includes DNA opening and is the rate-determining step of open complex formation.