Studies of the control of transcription of Xenopus 5S RNA genes have established this gene system as a leading model for understanding the regulation of transcription in eukaryotes. The 5S RNA gene is activated by the binding of a transcription factor, designated TFIIIA, to the enter of the 120 nucleotide long gene. This transcription factor, which is the prototype for a zinc-finger DNA binding protein, has the novel ability to bind specifically to 5S RNA as well as to 5S DNA. Experiments are proposed to further study the detailed structure of the 7S particle containing 5S RNA and TFIIIA. Preliminary results have led to a model for 7S particle structure that will be directly tested in these experiments. Binding of TFIIIA to the gene is only the first step in transcriptional activation. Transcription factor TFIIIC binds to the factor A-DNA complex to form a stable complex that resists competition by a second template. Further purification of Xenopus TFIIIC will be conducted to determine the extent to which its two separable components structurally and functionally resemble the human TFIIIC1 and TFIIIC2. DNase protection experiments will be conducted to determine whether the Xenopus TFIIIC2 binds to the site of transcription initiation, as has been suggested for its human counterpart. A collection of mutant 5S RNA genes with altered spacing between the TFIIIa binding site and the initiation site (containing the presumed TFIIIC2 binding site) will be studied to correlate transcription factor binding and DNAse protection stable complex. A novel protease-protection """"""""footprinting"""""""" technique will be developed in the stable complex. Three series of experiments are planned to study the role of RNA polymerase III. First, a chemical labeling procedure will be adapted to identify the polypeptides in the active site of the RNA polymerase in both the initiation and early elongation modes of transcription. Second, the significance of an active protein kinase in RNA polymerase III will be investigated. Finally, the question of whether RNA polymerase II is capable of accurate termination without the action of La antigen as a termination factor will be reinvestigated.
