(from the application): PolyA-dependent termination is a process that is orchestrated by the polyA signal and that involves multiple transcription and RNA processing components within a large rnRNA """"""""factory."""""""" The signal to terminate is generated during the assembly of the cleavage and polyadenylation apparatus. Using cis-antisense rescue for measuring the rate of 3'-end processing complex assembly in vivo we will ask: 1) Does assembly rate govern polyA site strength? and 2) Does assembly occur by stepwise addition of factors, or via a holocomplex? The ability of the polyA signal in the RNA to direct termination requires that 3'-end processing events be coupled to transcriptional events. How coupling is achieved is not known. We propose a model that is consistent with all available information. The main premise of the model is that coupling is achieved through intimate involvement of the polymerase with the assembly of the cleavage and polyadenylation apparatus. The model predicts unusual interactions between polyA sites located close to each other in vivo. For example, the total cleavage and polyadenylation activity for two strong polyA sites in tandem should decrease if the first site is weakened by mutation, but then increase again if the first site is completely inactivated. On the other hand, the model predicts that polyA sites will have no effect on one another when allowed to undergo processing in the absence of transcription in vitro. Experiments are proposed to test the various predictions of the model. Nothing is known of the molecular events involved in polyA-dependent termination because no system has yet been developed for studying polyA-dependent termination in vitro. We have now developed such a system and propose experiments to answer the following questions: 1) Is the processing factor, CPSF, required for polyA-dependent termination? 2) Is the transcription factor, TFIID, required to recruit CPSF? 3) Is termination mechanistically coupled to extrusion of the polyA signal? 4) How far must the polyA signal be extruded in order to function in driving termination? 5) At what point does the transcription elongation complex acquire termination competence? 6) At what point does the complex acquire the factors uniquely required for termination? Finally, methods for identifying specific factors involved in polyA-dependent termination are proposed. First, various direct approaches are presented for isolating termination factors from crude extracts. Then the possibility is considered of using immunodepletion to identify polyA-dependent termination factors from among proteins that have already been described.
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