Transcriptional enhancers are a crucial element in eukaryotic gene expression, yet their mechanism of action is largely unknown. The Xenopus rRNA enhancer is an excellent system in which to probe enhancer mechanism because it can be easily assayed by injection into oocytes and appears to require only a small number of proteins for its activity. When the rRNA enhancer and promoter are located in trans on the opposite rings of a multiply-intertwined dimeric catenane, the enhancer augments transcription. This result strongly suggests that enhancers work by looping, and this approach can be used to further test enhancer mechanism and to better understand transcription mechanisms. To test the importance of catenane structure in trans activation, gyrase network of the promoter and enhancer, which consist of single interlinks in essentially all combinations, will be prepared and assayed for enhancer activity. The importance of enhancer topology will be tested by linearizing the enhancer after transcriptional activation. The kinetics of transcription complex formation will be examined by oocyte injection. Oligonucleotide-directed triple helix formation will be tested for its usefulness as a tool in dissecting transcriptional mechanisms. The mechanisms of enhancer action and transcription of RNA polymerase II genes in Xenopus oocytes and embryos will be investigated by defining the regulatory sequences of oocyte-specific genes and testing trans activation of an embryonic enhancer. In a complementary approach, the interaction of the transcription factor THIS with both the promoter and enhancer will be characterized in more detail. The DNasel footprint of THIS suggests that the DNA wraps around the protein. This hypothesis will be tested by determining whether a change in the linking number of a plasmid containing THIS binding site in the circular permutation assay. Enhancer mutants that have changes either in spacing between sites or promoter THIS binding sites substitution for enhancer sites will be assayed for transcription. The importance of primary sequences and DNA structure in THIS binding and bending will be explored by combining DMS protection and interference, ethylation interference and bending studies with site directed mutagenesis of the THIS binding sites. the polypeptides copurifying with THIS activity will be characterized in more detail.
