The cellular process of transcription is fundamental to the well-being and development of all living things. We will characterize this process as it occurs in native replicated chromatin substrates to gain insight into mechanisms involved in activation and propagation of mammalian transcription. Native transcription complexes from transfected plasmid DNA as well as from simian virus 40 will be investigated. Two general aspects will be studied. First, the presence and location of transcriptional factors will be determined in the complex. The occupancy of a factor's DNA binding site will be determined using novel footprinting methods that are specific to the native transcription complex. Then identity of the transacting factor responsible for the footprint will be determined using imunoprecipitation. For simian virus 40 transcription complexes, two forms will be analyzed: the early mRNA-producing complex and the late mRNA-producing complex. Differences in the factor compositions of the transcriptional regulatory regions of the two types of complex will reveal the structural reconfiguration needed to cause the temporal shift from the early transcription unit to the late transcription unit. The analysis will also identify factors that commit the template to transcription. A second feature of native mammalian trancription complexes to be investigated will be the properties of the chromatin itself. The overall goal is to determine those structural properties that distinguish transcriptionally active from inactive chromatin. Due to recent findings that attribute an important transcriptional role to histone hyperacetylation, a particular focus will be the structural and functional consequences of histone hyperacetylation in native transcription elongation, on minichromosomal DNA topology, and on the interaction of histone H1 with the histone core.
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