The long-term objective of this proposal is to define the mechanisms by which transcription and its regulation are accomplished. These processes underlie basic functions such as morphogenesis and differentiation and are aberrant in cancer cells. As such, the knowledge is fundamental for understanding oncogenesis and oncogenic progression. RNA Polymerase II interacts with many proteins that regulate its function and is the core of the transcription mechanism. During elongation of the mRNA transcript, RNA polymerase II encounters """"""""pause"""""""" DNA sequences that appear in at least 40 percent of human open reading frames, causing the enzyme to arrest and preventing completion of mRNA synthesis. The regulatory protein TFIIS binds RNA Polymerase II and plays a crucial role in allowing RNA Polymerase II to complete mRNA synthesis. TFIIS, implicated in tumorigenesis, has a pivotal role in the regulation of transcription.
The specific aims of this proposal include; structure determination of yeast RNA Polymerase IITFIIS co-crystals, RNA Polymerase II structure-function relationships and the structure of mammalian RNA Polymerase II. The methodology employed to accomplish these goals involves the purification to homogeneity of yeast and mammalian RNA Polymerase II, as well as yeast TFIIS using ion exchange and affmity chromatography. Upon purification of the proteins, crystals are grown, diffracted in a synchrotron X-ray beam, and the structure determined. Site directed mutagenesis of RNA Polymerase II in yeast will allow for verification and determination of the structure-function relationships of the proteins.
