SPECTROSCOPIC DETERMINATION OF OPEN COMPLEX-FORMATION AT PROMOTERS FOR ESCHERICHIA-COLI RNA-POLYMERASE

A considerable amount of effort has been expended studying the kinetic s of association of Escherichia coli RNA polymerase with promoter DNA in forming the open complex. Strand separation occurs over about 12 ba se pairs and includes the transcription start site. However, these eff orts have been significantly hampered by the lack of a sensitive, real time method by which formation of an open complex could be assayed. H ere, we employ short (86 bp) synthetic promoters with 2-aminopurine (2 -AP) substitutions in the region that becomes single-stranded to spect roscopically monitor open complex formation. We demonstrate that promo ters bearing the substitutions behave in a manner similar to that of t hose containing only the four common bases with respect to both the re gion of strand separation and start site selection. Open complex forma tion was found to yield an increased fluorescence signal with an emiss ion maximum characteristic of 2-aminopurine. This spectroscopic assay for open complex formation was found to be well-suited to the investig ation of a strong promoter, allowing open complex formation to be foll owed over a time scale of seconds with a stopped flow apparatus. The i ntroduction of two additional nonconsensus base pairs in the -35 regio n resulted in a promoter for which open complex formation was 100-fold slower. The same substrates were also used to monitor the promoter re -annealing that ensues upon initiation of RNA synthesis. Similar rates for this process were observed for the two promoter variants employed in this study.