By selectively inhibiting the subunits of gyrase with coumermycin of oxolinic acid (or potentially with temperature shifts using ts gyrase mutants), we can alter the topology of chromosomal DNA in a defined way. In the coming year we will continue to explore the relationships between supercoiling, gyrase, and chromosomal functions. We intend to determine if gyrase activity occurs at specific or random chromosomal sites. We will radioactively label the end of DNA fragments produced by oxolinic acid-sodium dodecyl sulfate treatments and then determined by restriction nuclease analysis if these labeled ends are on specific or random fragments. We will also ask if the DNA relaxing activity seen after coumermycin treatment arises from the nicking-closing activity of gyrase. If so, we expect relaxation to be blocked at nonpermissive temperatures when coumermycin is added to cells carrying a ts mutation in the nicking-closing protein. A third goal is to measure how changes in DNA supercoiling (induced by coumermycin) affect binding of proteins to the chromosome. These studies will initially focus on RNA polymerase, and they may provide insights into the effect of supercoiling on transcription.