This is a competitive renewal application for a grant which has supported our efforts toward understanding the mechanisms of assembly of the transcription complex of the Xenopus 5S RNA genes and the transcriptional regulation of these genes during development. The proposed studies will focus on the trans-acting protein factors responsible for promoter binding and transcription initiation, modulation of transcription by post- synthetic modifications of the transcription factors, and differential transcription of the oocyte- and somatic-type 5S gene families. We plan to probe the interactions of the basal pol III transcription factors, TFIIIA, B and C, with one another and with the DNA template. A combination of biochemical and biophysical approaches to understand these interactions on both the oocyte- and somatic-type 5S genes will be utilized. We will map the sites of interaction of the polypeptide constituents of TFIIIB and TFIIIC with the 5S RNA genes by UV-crosslinking to site-specific photosensitive DNA probes. The identity and mechanism of action of a transcription stimulatory activity will be investigated. Recent studies in our lab have shown that the mitotic kinase, p34cdc2-cyclin B, can phosphorylate a component of the transcription apparatus and thereby repress pol III transcript ion. We will identify the substrate of mitotic phosphorylation responsible for this repression of transcription and obtain a cDNA clone for this protein(s). A bacterially-expressed protein will be used to map the amino acids phosphorylated by cdc2 kinase and mutagenesis experiments will be performed to provide unequivocal proof of the identity of the target of mitotic phosphorylation. The distribution of transcription complex components in chromatin will be investigated by a combination of protein-DNA crosslinking, immunofractionation and PCR amplification for detection of the crosslinked DNA. These studies will assess the role of specific components of the transcription apparatus in the activation and repression of the 5S RNA genes in living cells.
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