The role of DNA sequence in the recognition of transcription initiation sites (promoters) by RNA polymerase (RNAP) is being investigated by a combination of biochemical, genetic, and molecular biological techniques. Our primary interest is in characterizing interactions between RNA polymerase and the promoter, between specific activator proteins required for maximal transcription initiation and their DNA recognition sites, and between activator proteins and RNA polymerase. Mutations in promoters are analyzed for their effects on each of these aspects of promoter function in vitro and in vivo.
The main objects of this research are three particular promoters: 1) the PRE promoter of bacteriophage l, which is activated by the product of the phage gene cII; 2) the trpBA promoter of Pseudomonas aeruginosa, which is activated by the product of the trpI gene; 3) l PR, which is a strong, unregulated promoter. We demonstrated that activation of the trpBA promoter and l PRE both require the C-terminal portion of the a subunit of RNAP and have preliminary evidence that activation of PRE also requires wild-type amino acid sequences in the C-terminus of the RNAP s subunit. We are using structural studies and mutational analysis to study the interaction(s) of these activators with RNAP in greater detail.
We recently have investigated the effects of promoter mutations on the kinetics of transcription initiation. One set of mutations changes the spacing between two consensus hexameric DNA sequences recognized by RNA polymerase from 17 base pairs to 16 or 18 base pairs. Another set changes a base pair at position -10 in the corresponding consensus hexamer from T:A to G:C, C:G, or A:T. All of the mutations were shown to affect steps in transcription initiation that precede separation of DNA strands at the transcription start site. Most likely, the mutations affect an isomerization step that is thought to "nucleate" strand separation. The results with the single base pair substitution mutants demonstrate that the RNA polymerase s subunit recognizes the -10 base pair while the DNA is still double-stranded.