Differences in kinetics of transcription initiation by RNA polymerase (RNAP) at different promoters tailor the pattern of gene expression to cellular needs. After initial binding, large conformational changes occur in promoter DNA and RNAP to form initiation-capable complexes. To understand the mechanism and regulation of transcription initiation, the nature and sequence of these conformational changes must be determined. Escherichia coli RNAP uses binding free energy to unwind and separate 13 base pairs of λPR promoter DNA to form the unstable open intermediate I2, which rapidly converts to much more stable open complexes (I3, RPo). Conversion of I2 to RPo involves folding/assembly of several mobile RNAP domains on downstream duplex DNA. Here, we investigate effects of a 42-residue deletion in the mobile β′ jaw (δJAW) and truncation of promoter DNA beyond +12 (DT+12) on the steps of initiation. We find that in stable δJAW open complexes the downstream boundary of hydroxyl radical protection shortens by 5-10 base pairs, as compared to wild-type (WT) complexes. Dissociation kinetics of open complexes formed with δJAW RNAP and/or DT+12 DNA resemble those deduced for the structurally uncharacterized intermediate I3. Overall rate constants (k a) for promoter binding and DNA opening by δJAW RNAP are much smaller than for WT RNAP. Values of ka for WT RNAP with DT+12 and full-length λPR are similar, though contributions of binding and isomerization steps differ. Hence, the jaw plays major roles both early and late in RPo formation, while downstream DNA functions primarily as the assembly platform after DNA opening.