Structural-Function Studies of Shared hRNAP Components

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DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the mechanisms of the transcriptional regulation of gent expression at the molecular level. Transcription of all eukaryotic nuclear genes is performed by three distinct RNI polymerases (RPs) I, II, and III, which synthesize ribosomal, messenger, and transfer RNAs, respectively. RPs are multi-subunit complexes, consisting of enzyme-specific polypeptides and subunits common to all three enzymes. The human common subunits (hRPB5, hRPB6, hRPB8, hRPB10-alpha, and hRPB10-beta) are bona fide universal transcription factors because they are essential for the expression of all human genes by the three hRPs. Therefore, determining the structure and function of these subunits is an essential step towards elucidating the basic molecular mechanisms that control global human gene transcription. The principal investigator has already determined the solution structure o hRPB6 using Nuclear Magnetic Resonance (NMR) spectroscopy. The present proposal focuses on the structural analysis of the common human subunits and the elucidation of their roles in the molecular architecture and function of human RPII. The specific aims and experimental approaches of the proposed research are: 1) To perform a high-resolution structural analysis of the common subunits hRPB8, hRPB10-alpha, hRPB10-beta, using X-ray crystallography and NMR spectroscopy. 2) To determine the structural basis for the interactions between hRP common subunits and general transcription initiation and elongation factors. In this aim it is proposed to perform an X-ray crystallographic analysis of the hRPB5 subunit in complex with transcription initiation factor hTFIIB and of the hRPB6 subunit in complex with the transcription elongation factor hTFIIS. 3) To dissect the role of the common subunits hRPB10alpha, hRPB10beta, and hRPB8 in the function of tumor suppressor proteins. These studies will provide high-resolution three-dimensional structures of the human common subunits and will elucidate their roles in the assembly, structure, and function of the three nuclear hRPs. The atomic coordinates of the common subunits, besides their scientific value, could also be used for structure-based rational design of chemicals that could modify selectively the structure and function of the hRPs, with potential applications in molecular medicine.