The 5S RNA genes of the African frog, Xenopus, provide a model system in which to study the regulation of transcription of eukaryotic genes. Techniques such as in vitro transcription and deletion mutagenesis first established in this gene system have stimulated research on a large variety of other cloned genes. The first step in activation of a 5S RNA gene for transcription is the binding of a positive factor, designated TFIIIA to the center of the gene. Experiments will be performed to test a recently devised model that predicts a colinear binding of this protein along the 5S DNA. This will involve construction of a compensatory mutation in the TFIIIA protein that is predicted to rescue a mutant 5S RNA gene with a deletion in the center of the intragenic control region. A novel indirect footprinting procedure has been developed in our laboratory and used to show that this protein binds as well to the major oocyte type 5S RNA genes as to the somatic type 5S RNA genes. It appears that the relatively poor ability of the major oocyte genes to compete for active transcription complex formation results from a defect in a subsequent step in transcription complex formation, such as the binding of transcription factor TFIIIC to the TFIIIA:5S DNA complex. Experiments are proposed to study the stages in active transcription complex formation that may be defective in both the oocyte gene and point mutants in the somatic 5S RNA gene. These variant gene sequences will also provide a means to study the role of chromatin structure in the activation of genes for transcription. It is of particular interest to determine whether the type II topoisomerase plays a role in formation of active transcription complexes.
