Diana K. Hawley
Regulation of Mammalian RNA Polymerase IIMy group is interested in the enzymology of RNA polymerases and the mechanisms by which eukaryotic transcription is regulated. The overall goal of our research is to identify and characterize proteins that determine the transcription initiation and elongation properties of mammalian RNA polymerase II, the enzyme responsible for synthesis of messenger RNA in vivo. The overall strategy is first to dissect the complex process of transcription into a series of quantifiable functional steps and then to determine which of these steps are relatively invariant and which are modulated by DNA sequences or regulated by transcriptional activator or repressor proteins.
Research from a number of laboratories has identified at least six proteins from human cells that are required, in addition to RNA polymerase II, to reconstitute promoter.dependent transcription in vitro. These proteins appear to assemble at the promoter in an ordered fashion prior to transcription initiation. The details of this assembly and the specific function of individual proteins in the initiation process are being addressed by my laboratory group. Current research is focused on protein.DNA complexes that appear to be functional intermediates during the early steps of the assembly pathway. The group is using a variety of biochemical and biophysical methods to measure the contributions of DNA sequence and protein.protein interactions to the rate of formation and stability of these various complexes. In addition, we have undertaken collaborations with colleagues in the Department of Chemistry and the Institute of Molecular Biology to probe the structure of these complexes by other physical methods including the direct visualization of these complexes by scanning force microscopy.
Although eukaryotic transcription appears to be regulated mainly at the level of initiation, recent studies have suggested that premature termination of transcription at specific sites is also an important mechanism for the regulation of expression of some protein.encoding genes in vivo. My group is studying the mechanism of transcription arrest at one such site within a human viral genome and has begun to define the DNA sequences and cellular proteins that influence the extent of readthrough at this site. One particularly interesting finding that emerged from this study is that the efficiency with which transcription elongation is blocked at this site is dependent on the promoter from which the transcript initiates. An important aim of this research is to understand the biochemical basis of this promoter specificity. Another active area of research that has opened up through this study is based on the discovery that RNA polymerase II has a 3U to 5U exonuclease activity. This activity is greatly stimulated by a protein called SII, known to aid transcription elongation by promoting readthrough of arrest sites. The relationship between the exonuclease activity and increased readthrough is being investigated in the laboratory. In addition, other possib