The long-range goal of the research is to detail the events that occur during the initiation of transcription of protein coding genes (also called II genes). This will be accomplished through a thorough characterization of the proteins involved and the various protein:DNA and protein:protein interactions. The overall aim of this proposal is to continue and expand studies now in progress in the laboratory on the identification and characterization of the protein factors which are required for basal levels of transcription (general transcription factors GTFs), and to elucidate the molecular mechanisms that govern promoter recognition by RNA polymerase II. These studies will include analyses of how gene specific transcription factors (activators) modulate the activity of the general transcription factors.
The specific aims during the tenure of this application will be to obtain cDNA clones to the general transcription factors as well as the factors required to enhance the response to activators. The investigator plans a detailed study of the reactions catalyzed by the general transcription factors, including analyses of how specific factors modify the activity of the general transcription factors, RNA polymerase II and promoter sequences through the use of techniques such as glycerol gradient centrifugation, gel filtration, immunoprecipitation and protein cross linking. DNA footprinting will be used to analyze the DNA-protein interaction. Kinetic analysis of the association of the different factors with the preinitiation complex will be performed in the presence and absence of activators, to understand how gene specific transcription factors modify the assembly of the general transcription factors with the preinitiation complex. The studies may yield basic model(s) from which the specific regulatory mechanisms of class II gene expression can be discerned at the molecular level.
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