The objective of this proposal is to define the components of intermediate structures in the initiation of eukaryotic transcription and to study interactions of the components as revealed by geometrical relationships, binding dependencies and order of assembly. The model of investigation is to adapt biochemical methods to the requirements of Scanning Transmission Electron Microscopy (STEM).
Specific aims i nclude: in adenvirus 2, analysis of the joint binding of the protein factors USF and TFIID in the initiation of transcription at the major late promoter; in SV40, study of the individual structures and the interactions of initiation factor Sp1 and the enhancer-specific proteins AP1 and AP2. In these systems and in related cellular promoters we will seek to determine whether a variable sequence separation between promoter elements is nonetheless consistent with contact protein-protein interactions. The location of purified eukaryotic RNA polymerase II will be observed relative to partial assemblies of initiation factors at the major late promoter, with the view to determining which factor of factors lead to specific binding. Mass distributions of the 5S RNA initiation factor TFIIIA will be used to determine the number of monomer units bound at the control region and to test developing models for the protein's structure. Means will be sought to link gel-shift (co-electrophoretic) enrichment of DNA protein assemblies and STEM, in an effort to provide digital images of structures contained within well-defined bands on a gel.
