This is a proposal to continue studies of eukaryotic ribosomal RNA transcription initiation and regulation. RNA polymerase I (pol I) transcribes this gene. The rRNA gene of the eukaryotic organism being studied, Acanthamoeba, has properties making it especially good for the biochemical studies proposed; properties no other pol I system currently exhibits. In particular, the unusual stability of the complex between its core promoter and transcription factor allow analysis by techniques not available in other systems. Recent findings lead logically into this proposal. The entire rRNA intergenic spacer was sequenced, pol I enhancers were identified, and shown to bind a vertebrate UBF homolog. The TATA-binding protein (TBP)- containing initiation factor (TIF-IB) was purified to homogeneity, its subunits (TAFIS) identified, and TBP's distinctive role in pol I transcription revealed. NTP beta-gamma hydrolysis was shown not to be needed for initiation, and the DNA melting process by pol I was examined in detail. Site-direct photocross-linking of TIF-IB and pol I to promoter DNA resulted in mapping the topology of TAFIS and TBP on the rRNA promoter. Coupled with STEM pictures, this study has given insights into the activated promoter complex never before detailed, and gave significant insights into how pol I promoters function. Regulatory studies implicated the pol alpha subunit homolog (AC39) in regulation, and inroads into its cloning and sequencing were made. Funds are now requested to attain the following aims: (A) Protein-protein and protein- DNA interaction will further detailed using new sophisticated photoaffinity probes, heterobifunctionals cross-linking agents, and high resolution STEM. (B) The role, if any, of several potential additional transcription factors which influence rRNA transcription will be evaluated. These include a Ku-like factor and a factor required for stable complex formation on the core promoter. (C) The regulatory mechanism of rRNA transcription will be investigated, placing emphasis on the chemical events resulting in the inactivity of pol I purified from transcriptionally inactive cells. Investigation of the known modification of the AC39 subunits, and its role in regulation, will be a particular aim. (D) Genes for the TAFIS and key pol I subunits will be cloned. (E) A method for transient and stable transformation of Acanthamoeba will be developed.
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