My group is interested in understanding specific chemical interactions which underlie complex enzyme systems. A major focus of the lab is to understand structure and function in enzyme-nucleic acid interactions. In transcription, the enzyme RNA polymerase must not only bind specifically to its promoter DNA sequence but must then initiate the processive catalysis of template-dependent RNA synthesis, leave the initial recognition site (promoter), and continue on to a stable, sequence-independent complex.
The T7 family of RNA polymerases present an ideal model system for the study of transcription. The single subunit enzyme is relatively simple and can be readily purified in large quantities from overproducing strains of E. coli. The chemical synthesis of oligonucleotides containing specifically modified base substituents, has allowed us to map out functional groups on the DNA which are critical to recognition and initiation. We can, for example, remove a single hydrogen bond donor or acceptor from a known position in the DNA helix and then ask quantitatively how recognition is perturbed. We can also introduce fluorescent base analogs that report on the melted state of the DNA, in order to map movement of the transcription bubble.
Recent crystal structures of initiation and elongation complexes (with DNA and RNA) provide a wealth of testable models for various aspects of this complex chemical process. Stopped flow and quench flow kinetic studies have allowed a dissection of the mechanistic steps in the initiation of transcription, while site-directed mutagenesis has allowed us to probe possible structural interactions. Our ultimate goal is to directly link changing structural interactions between the enzyme and DNA to individual chemical events in the transcription process.