Transcriptional regulation is a central mechanism for the control of gene expression in both eukaryotic and prokaryotic cells. While control at the level of transcriptional initiation is an important process, significant regulation also occurs at the elongation and termination phases of transcription. Recent studies suggest that termination/antitermination signals are important in differentiation and in expression of cellular oncogenes. However the processes of transcription elongation and termination are poorly defined for eukaryotic cells, and the there is little known about the molecular mechanisms involved. The rapid and extensive processing of eukaryotic transcripts, taken with the complexities of defining biochemical events in isolated cell nuclei and cell extracts, make it difficult to define true termination sites, or termination factors, in vivo, or in an undefined in vitro system. A plausible solution to defining the sites and components that specify termination for RNA polymerase II involves development of an in vitro transcription system with purified components in which true termination events can be studied and distinguished unequivocally form processing events. We have developed a system for study of termination and elongation events using defined linear DNA templates bearing poly dC tails that are efficiently transcribed by highly purified eukaryotic RNA polymerases. In transcriptional studies of well characterized prokaryotic and eukaryotic sequences by calf thymus RNA polymerase II, we have shown that RNA polymerase II recognizes certain CNA regions as intrinsic termination signals, at which chain termination and RNA release both occur. Some intrinsic termination signals are located within eukaryotic genes, suggesting the possibility of efficient antitermination mechanisms. We propose to continue our studies of elongation and termination reactions carried out by mammalian RNA polymerase II, with particular emphasis on the structure of the intrinsic DNA termination signals used by these enzymes, and the factors that enhance or suppress termination at such sites.
Specific aims of these studies include: 1) Identify the DNA sequences that make up the intrinsic termination signal for purified RNA polymerase II, construct mutant termination sites to determine the effect of such changes on termination by RNA polymerase II in vivo and in vitro, and compare termination site recognition properties of other eukaryotic RNA polymerases. 2) Identify, purify and characterize protein factors that affect the use of termination signals by RNA polymerase II including possible antitermination and termination factors. 3) Characterize the structure and composition of ternary elongation complexes between highly purified RNA polymerase II, the template, and the transcript, when the enzyme is stopped at specific sequences. 4) Characterize the elongation and termination reactions of purified RNA polymerase II with nucleoprotein templates to determine the basic features of elongation and termination through chromatin DNA sequences.
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