Abstract
A quantitative kinetic mechanism for transcription initiation at the bacterial promoter glnAp2 was previously determined using single molecule experiments with relaxed DNA. DNA in E. Coli cells, however, is rarely relaxed but is instead negatively supercoiled. Bulk experiments in the literature report a 10 to 60 times increase in the overall rate of transcription initiation at the glnAp2 promoter with negatively supercoiled DNA compared to relaxed DNA. To locate where in the mechanism this increase in rate occurs, I created circular DNA that contained the glnAp2 promoter, biotin for attachment to a microscope slide, and a dye for visualization. Initiation was then measured using single molecule techniques on both nicked and negatively supercoiled forms of this template. The overall rate of transcription initiation was seven times faster on negatively supercoiled DNA compared to on nicked DNA. The rate of RNA polymerase binding, however, was only 1.5 times faster on supercoiled DNA, and the rate of open complex decay on the supercoiled and nicked templates was not significantly different. Initial experiments on the RNA polymerase closed complexes suggest that their stability on negatively supercoiled and nicked templates is not very different. Based on those results, I hypothesize that isomerization from closed to open complex is most likely the step in the mechanism that accounts for the difference in overall rate of transcription initiation between relaxed and supercoiled DNA.