Genes are composed of DNA and contain the information encoding proteins in cells. In eukaryotes, this information is transcribed from DNA into RNA by an enzyme called RNA polymerase II. Much is known about how RNA polymerase II associates with the beginning of a gene to initiate transcription but little is known about how RNA polymerase II disengages from the DNA after reaching the end of a gene. This disengagement is known as termination. This project investigates the mechanism by which RNA polymerase II terminates transcription at the end of a gene. The protein, Pcf11, was found to disengage RNA polymerase II from DNA by a mechanism in which Pcf11 forms a bridge between the RNA being synthesized and a large protrusion on the RNA polymerase II known as the CTD. To elucidate the mechanism of this reaction, the effect of purified Pcf11 on purified RNA polymerase II engaged in transcription will be analyzed. Pcf11 was also found to associate with the end of a heat shock gene in live Drosophila cells, and depletion of Pcf11 from the cells inhibited the ability of RNA polymerase II to disengage from the heat shock gene. To determine how broadly Pcf11 functions in termination, this project will investigate the role of Pcf11 in causing termination on numerous other genes in Drosophila cells. Finally, specific sequences in the transcript are known to contribute to the termination mechanism. In contrast, isolated Pcf11 appears to cause termination indiscriminately. To determine the basis for the sequence specificity of termination, the ability of Pcf11 in association with other cellular factors to cause termination will be investigated. In addition, another protein from yeast called Nrd1 will be evaluated for termination activity, since this protein is predicted to have the ability to bridge the CTD to the RNA in a fashion dependent on the RNA sequence. Transcription termination is essential for the viability of cells, and mutations in Pcf11 in yeast and in Drosophila are lethal. The disengagement of RNA polymerase II at the ends of genes is essential to reuse the polymerase and to prevent polymerase molecules from disrupting the transcriptional activity of adjacent genes. This project should provide significant insight into this fundamental cellular process. The project also provides an excellent basis for training students and postdoctoral scientists in molecular biology. The combination of biochemical and cellular approaches will provide individuals with skills needed to investigate gene regulation in living cells.
