This is a proposal to study the mechanism of rRNA transcription and its regulation in eukaryotes. An in vitro transcription system made up of cloned rDNA, homogeneous RNA polymerase I (RNAP I) and an auxilliary protein (transcription initiation factor(s)-TIF) is being utilized: (1) to determine the number of TIFs, and to purify them to homogeneity, (2) to determine in greater detail the role played by the TIF in initiation of transcription, (3) to determine how the TIF and RNAP I interact with each other and with the template and (4) to describe kinetically the events involved in the assembly of the initiation complex. DNA footprinting and chemical modification-protection experiments will be used to determine protein-DNA contacts. Chemical cross-linking experiments will be used to determine protein-protein interactions. Kinetic measurements will be used to determine the kinetic mechanism of initiation. Monoclonal antibodies to the RNAP I subunits and the TIF(s) will be produced to aid in the analysis. Transcription of rRNA gene expression is developmentally regulated in Acanthamoeba by modification of RNAP I. Experiments to identify the chemical nature of the modification and the enzyme catalyzing it are proposed. Exactly which step in the process of initiation is affected by the modification is also to be studied utilizing the techniques proposed to study the mechanism of initiation.
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