Kinetic-Mechanistic Evidence for Which E. coli RNA Polymerase-{lambda}PR Open Promoter Complex Initiates and for Stepwise Disruption of Contacts in Bubble Collapse

In transcription initiation, specific contacts between RNA polymerase (RNAP) and promoter DNA are disrupted as the RNA-DNA hybrid advances into the cleft, resulting in escape of RNAP. From the pattern of large and small rate constants for steps of initiation at {lambda}PR promoter at 19{degrees}C, we proposed that in-cleft interactions are disrupted in extending 3-mer to 5-mer RNA, -10 interactions are disrupted in extending 6-mer to 9-mer, and -35 interactions are disrupted in extending 10-mer to 11-mer, allowing RNAP to escape. Here we test this mechanism and determine enthalpic and entropic activation barriers of all steps from kinetic measurements at 25{degrees}C and 37{degrees}C. Initiation at 37{degrees}C differs significantly from expectations based on lower-temperature results. At low concentration of the second iNTP (UTP), synthesis of full-length RNA at 37{degrees}C is slower than at 25{degrees}C and no transient short RNA intermediates are observed, indicating a UTP-dependent bottleneck step early in the 37{degrees}C mechanism. Analysis reveals that the 37{degrees}C {lambda}PR OC (RPO) cannot initiate and must change conformation to a less-stable initiation complex (IC) capable of binding the iNTP. We find that IC is the primary {lambda}PR OC species below 25{degrees}C, and therefore conclude that IC must be the I3 intermediate in RPO formation. Surprisingly, Arrhenius activation energy barriers to five steps where RNAP-promoter in-cleft and -10 contacts are disrupted are much smaller than for other steps, including a negative barrier for the last of these steps. We interpret these striking effects as enthalpically-favorable, entropically-unfavorable, stepwise bubble collapse accompanying disruption of RNAP contacts. SignificanceTranscription initiation is highly regulated. To understand regulation, mechanisms of initiation and escape of RNA polymerase (RNAP) from the promoter must be understood. RNAP forms a highly-stable open complex (RPO) with {lambda}PR promoter at 37{degrees}C. From experiments determining effects of temperature on rate constants for each step of RNA synthesis, we find that RPO cannot bind the initiating nucleotides, that the I3 intermediate and not RPO is the initiation complex, and that contacts of RNAP with single-stranded DNA of the discriminator and -10 region and with -35 duplex DNA are disrupted stepwise as the RNA-DNA hybrid moves into the cleft. Evidence is obtained for stepwise bubble collapse and base stacking accompanying disruption of interactions of the single-stranded discriminator and -10 regions with RNAP.