Bacteria need to alter their transcriptome quickly to respond to changes in their environment. In E. coli, the second messenger ppGpp and the transcription factor DksA regulate over 750 genes to help cells respond to various nutritional stresses. Neither ppGpp nor DksA bind to DNA. Instead, they act directly on RNAP, allosterically activating or inhibiting transcription of different genes. Understanding the conformational changes resulting from DksA and ppGpp interactions with RNAP is key to understanding the mechanism of regulation. I studied the ability of various RNAP variants to respond to DksA and ppGpp. Multiple mobile elements of RNAP are needed for activation and regulation by DksA and ppGpp, including the β’ clamp, the trigger loop (TL), and β sequence insertion 1 (βSI1). In collaboration with structural biologists Andrey Feklistov and Seth Darst at Rockefeller, we determined that DksA and ppGpp result in significant movements of these elements that correlate with effects of mutations on RNAP function. Interaction of DksA with the TL favors clamp closure to favor melting of the transcription bubble. Movement of βSI1 by DksA and ppGpp widens the cleft and alters contacts to downstream DNA. The role of mobile elements has previously been difficult to characterize because they are hard to capture in crystal structures. These data provide a model for how DksA and ppGpp activate or inhibit transcription at different promoters. I also characterized a transcription intermediate in which the transcription bubble is only partially melted. DNA footprinting and Bpa crosslinking data show that DksA/ppGpp can inhibit melting of the full transcription bubble at rpsT P2 but does not inhibit capture of the -11A base or melting of the upstream region. Furthermore, capture of the -11A base does not require DNA to be positioned near the active site within the cleft. Together, these data provide new insights into the mechanism of transcription initiation in bacteria and its regulation.