The objective of our research is to understand how regulatory proteins interact with DNA and therefore control gene expression. Our studies focus on two systems-the lac operon and the major rightward control region of bacteriophage lambda. Lac operators containing sequence and functional group modifications are being synthesized and biochemically analyzed. Similarly a large number of base analogs and sequence changes will be introduced into lambda PR. These substitutions will permit us to precisely map those functional groups on promoters that are recognized by E. coli RNA polymerase, lac, cI and cro repressors, and catabolite activator protein. The research so far suggests that lac repressor may not recognize a unique lac operator sequence. This research should also help define the steps involved in initiation of transcription, the mechanism of catabolite activation, and how two proteins (cro and cI) with quite different amino acid sequences recognize the same DNA sequence. Research will also be directed toward understanding the mechanism whereby catabolite activator protein stimulates transcription. Initially deletions and insertions will be introduced synthetically into the lac promoter at selected sites and the effect of these modifications will be measured in appropriate E. coli strains. Other research will focus on the amino acid residues in lac repressor and cro repressor that recognize specific DNA sequences. Lac repressor can be specifically cross linked with lac operator using bifunctional reagents. The crosslinking sites between DNA bases and amino acid side-chanis will be investigated. Cro repressor will be probed by site-specific mutagenesis. Amino acid changes will be introduced into cro by modifying the cro gene. The effect of these modifications on cro binding will be monitored in vivo and in vitro.
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