RNA Polymerase: Step-by-Step Kinetics and Mechanism of Transcription Initiation

Abstract

To determine the step-by-step kinetics and mechanism of transcription initiation and escape by E. coli RNA polymerase from the λPR promoter, we quantify the accumulation and decay of transient short RNA intermediates on the pathway to promoter escape and full-length (FL) RNA synthesis over a wide range of NTP concentrations by rapid-quench mixing and phosphorimager analysis of gel separations. Experiments are performed at 19 °C, where almost all short RNAs detected are intermediates in FL-RNA synthesis by productive complexes or end-products in nonproductive (stalled) initiation complexes, and not from abortive initiation. Analysis of productiveinitiation kinetic data yields composite second-order rate constants for all steps of NTP binding and hybrid extension up to the escape point (11-mer). The largest of these rate constants is for incorporation of UTP into the dinucleotide pppApU in a step which does not involve DNA opening or translocation. Subsequent steps, each of which begins with reversible translocation and DNA opening, are slower with rate constants that vary more than ten-fold, interpreted as effects of translocation stress on the translocation equilibrium constant. Rate constants for synthesis of 4-and 5-mer, 7-mer to 9-mer and 11-mer are particularly small, indicating that RNAP-promoter interactions are disrupted in these steps. These reductions in rate constants are consistent with the previously-determined 9 kcal cost of escape from λPR. Structural modeling and previous results indicate that the three groups of small rate constants correspond to sequential disruption of incleft, −10 and −35 interactions. Parallels to escape by T7 RNAP are discussed.

Kinetic-Mechanistic Evidence for WhichE. coliRNA Polymerase-λPROpen Promoter Complex Initiates and for Stepwise Disruption of Contacts in Bubble Collapse

“…Values of kI are much larger for synthesis of 3-mer, 6-mer, and 10-mer RNA than for the other six steps (32).…”

“…We previously proposed that the population distribution of λPR OC also changed with temperature, shifting from the very-stable 37 o C RPO with strong downstream interactions between RNAP DME and duplex DNA extending to +20 to a mixed population of RPO and the less stable intermediate OC designated I3 at lower temperature (25). If only I3 and not RPO can bind the two initiating NTP, then the differences in rates of FL RNA synthesis between (32). In addition to disruption of one DNA-DNA base pair, translocation stress accumulates and RNAP-promoter contacts are disrupted in many of these steps.…”

“…for the individual steps of NTP addition up to promoter escape (2 ≤ i ≤ 11) at 19 o C. Values of ki includes contributions from reversible translocation, reversible NTP binding and irreversible catalysis (32). Values of ki for steps of initiation involving translocation divide into three groups.…”

“…Elongation kinetic studies reveal that the translocation step before NTP binding is rapidly reversible and that the post-translocated state is intrinsically less stable than the pretranslocated state (33)(34)(35). The post-translocated state is stabilized by binding of the next NTP (32,35). In initiation, effects of translocation stress greatly increase the bias toward the pretranslocated state (32).…”

“…The post-translocated state is stabilized by binding of the next NTP (32,35). In initiation, effects of translocation stress greatly increase the bias toward the pretranslocated state (32). Large ki values indicate elongation steps where translocation is less unfavorable because stress has not yet built up (2-mer  3-mer) or where stress has been released by disruption of contacts in previous steps (5-mer  6-mer; 9-mer  10-mer).…”