The goal of this proposal is to contribute to the understanding of transcription initiation from the lactose (lac) operon of Escherichia coli. The first objective of the proposal is to obtain large quantities of two DNA fragments which contain the binding sites for RNA polymerase and the catabolite activator protein - cyclic adenosine monophosphate complex (CAP.cAMP). This will be done by growing up cells containing plasmids with the DNA fragments inserted into a single restriction endonuclease site, and cutting out and isolating the DNA fragments. These DNAs will be combined and altered to generate a modified promoter DNA with nucleotide analogues at specific locations near the transcription startpoint. Biophysical and Biochemical studies will be carried out on the DNAs. The influence of nucleotide replacements on RNA polymerase binding and transcription will be examined. Raman spectroscopy will be employed to examine the conformation of the unmodified lac DNA promoter and to determine the effect of CAP.cAMP binding on the vibrational properties of base pairs around the startpoint region.
