One of the basic points needed for understanding the molecular mechanisms involved in gene regulation and cellular differentiation is the process of how eukaryotic RNA polymerase interacts with the vast array of proteins assembled onto DNA to initiate transcription. This is an important area of health related research because of its relevance in helping to understand the molecular basis of many diseases such as AIDS and cancer. In trying to answer this question an innovative approach is proposed for analyzing the structure and function of protein domains in large nucleoprotein assemblies. Novel photoaffinity nucleotide analogs will be used for mapping the location of yeast RNA polymerase III subunits in the transcription complex. Mapping of the RNA polymerase III subunits (and peptide regions) will be in terms of distance from a particular nucleotide base or phosphodiester linkage in DNA. These photoaffinity nucleotide analogs are designed to have the unique features of a cleavable photocrosslinker that transfers radiolabel to protein and after photocrosslink cleavage the DNA is modified so that the photocrosslinking site in DNA can be identified. Locating the photoaffinity labeling site at the peptide level will be done by analysis of the pattern of peptide fragments generated by enzymatic or chemical degradation of the photoaffinity labeled subunit, and microsequencing of the unmodified peptide fragment corresponding to the photoaffinity label peptide. Additional structural information will be obtained using radiolabeled protein crosslinkers to (1) determine the placement of RNA polymerase subunits relative to each other and to transcription factor(s) in the fully assembled transcription complex, (2) assist in the determination of RNA polymerase III subunits contacting the initiation factor TFIIIB, and (3) identify TFIIIB and TFIIIC subunits that may not be in close proximity to DNA. In order to use the protein crosslinkers, the transcription complexes will have to be selectively attached to DNA using biotinylated DNA photoaffinity probes, and isolated away from free polymerase or polymerase nonspecifically bound to DNA or other contaminating protein. Structural information obtained in the described manner will be used in selecting peptide regions for site-directed mutagenesis of RNA polymerase III subunit(s) that are in close proximity to the transcription initiation factor or start site of transcription. The RNA polymerase mutants will be engineered to contain an affinity tag to allow for the synthesis of the mutated protein in the presence of functional wild type, in vivo assembly of the mutant protein, and the separation of the mutant RNA polymerase from wild type. Mutant RNA polymerase III will be characterized in a reconstituted in vitro transcription system for nonspecific transcription activity, binding to promoter DNA, formation of """"""""open"""""""" promoter complex, and promoter- dependent transcription.
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