This research proposal is directed towards extending our understanding of the organization and function of transcriptional control regions in chromatin. We plan to concentrate on the organization of a few specific systems such as SV40, Mo-MuLV and ASV. In order to study the organization of the transcriptional control regions of the viral genomes in chromatin form (particularly in terms of the generation of nuclease hypersensitive sites), we plan to reassemble chromatin from appropriate DNA (in circular form) and histones using an assembly system which we have developed based on original work done by Arnold Stein. We wish to assay if specific DNA sequences govern the placement and organization of nucleosomes on these DNA molecules, or if cellular protein factors are also involved. If the latter is the case we shall attempt to isolate and characterize these factors. We also wish to study initiation and chain elongation of RNA transcripts from these in vitro assembled nucleoproteins. Here we will pursue two general points (1) we will attempt to extend our initial observation that nucleoplasmin plays a key role (in vitro) in chain extension through nucleosomes and (2) we plan to study the initiation process on a defined chromatin template using partially purified transcription extracts capable of mediating accurate initiation on naked DNA templates.
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