This is an amended competitive renewal application for a grant which has supported the investigator's efforts toward understanding the developmental regulation and mechanisms of transcription of the Xenopus 5S RNA genes. The proposed studies will focus on the organization of the oocyte and somatic-type 5S RNA genes in chromatin, differential transcription of these genes in vitro, and the nature of the trans-acting protein factors responsible for promoter binding and transcription initiation. The investigator plans to probe the structure of the 5S gene promoter as protein-free DNA and to examine protein-induced changes in DNA conformation such as DNA bending. A combination of biochemical and immunological approaches have revealed different molecular forms of the 5S gene-specific transcription factor TFIIIA. These proteins will be studied with respect to primary structure, developmental regulation of mRNA and protein abundance, and mechanisms of protein binding to DNA promoter elements. These studies will include cDNA cloning and sequencing of the TFIIIA variant present in mature oocytes, embryos and somatic cells. The interactions of transcription factor TFIIIC with the TFIIIA-DNA complex on both the oocyte and somatic-type genes will be examined with respect to affinity constants and stability of the complexes. The DNA sequence elements responsible for TFIIIC interactions with 5S genes will be mapped through the use of substitution mutants, by DNase footprinting, and by methylation interference. A cDNA clone for the DNA-binding subunit of TFIIIC will be isolated and its primary sequence will be determined. Based on this information, the protein will be characterized with respect to functional domains involved in DNA biding and transcriptional activation. The in vivo distribution of transcription factor IIIA will be mapped by three techniques: In situ immunofluorescence on metaphase chromosomes, chromosomal footprinting, and protein-DNA crosslinking followed by immunofractionation. These studies will assess the role of TFIIIA in the activation and repression of the 5S gene families.
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