Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter. Evidence for a sequential mechanism involving three steps

The forward and reverse kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter have been studied in the temperature range of 15-42 degrees C. The standard two-step model, involving the formation of a closed intermediate, RPc, followed by an isomerization that leads to the active complex RPo, could not account for the present data. The promoter-enzyme lifetime measurements showed an inverse temperature dependence (apparent activation energy, -35 kcal/mol). A third step, which is very temperature dependent and which is very rapid at 37 degrees C, was postulated to involve the unstacking of DNA base pairs that immediately precedes open complex formation. Evidence for incorporating a new binary complex, RPi, in the pathway was provided by experiments that distinguished between stably bound species and active promoter after temperature-jump perturbations. These experiments allowed measurement of the rate of reequilibration between the stably bound species and determination of the corresponding equilibrium constant. They indicated that the third step became rate limiting below 20 degrees C; this prediction was checked by an analysis of the forward kinetics. A quantitative evaluation of the parameters involved in this three-step model is provided. Similar experiments were performed on a negatively supercoiled template: in this case the third equilibrium was driven toward formation of the open complex even at low temperature, and the corresponding step was not rate limiting.     

Structure of open promoter complexes with Escherichia coli RNA polymerase as revealed by the DNase I footprinting technique: compilation analysis.

Footprinting data for 33 open promoter complexes with Escherichia coli RNA polymerase, as well as 17 ternary complexes with different regulators, have been compiled using a computer program FUTPR. The typical and individual properties of their structural organization are analyzed. Promoters are subgrouped according to the extent of the polymerase contact area. A set of alternative sequence elements that could be responsible for RNA polymerase attachment in different promoter groups is suggested on the basis of their sequence homology near the hyperreactive sites. The model of alternative pathways used for promoter activation is discussed.     

Expression of the lac operon in RNA polymerase mutants of Escherichia coli K12.

Summary Radioactively labeled 4.5S, 6S, and 10S RNAs from Escherichia coli were hybridized to EcoRI fragments from the E. coli genome. Each of these molecules bound to more than one DNA fragment. C_ot curve analysis of the kinetics of the annealing of these RNAs to denatured E. coli DNA suggests that the DNA corresponding to each of these molecules is reiterated in the genome. These experiments also suggest that these reiterated sequences are non adjacent.