Gene expression is controlled in part by regulating the ability of RNA polymerase II to transcribe full length primary transcripts. Elongation is not simply the addition of single ribonucleotide units to a growing chain; instead, it is characterized by a striking plasticity in structural and functional alternatives that RNA polymerases can assume. Recent advances have identified new structural and functional intermediates that are the target of regulatory events. The transcription process can be blocked by specific DNA elements called arrest sites within genes and DNA binding ligands that interrupt elongation. Only a few arrest sites are known and the defining DNA sequence has not been resolved. Specific elongation factors improve the efficiency of RNA chain synthesis. Two such factors, TFIIF and SIII (elongin), are implicated in human diseases including cancer, demonstrating that perturbation of transcript polymerization is potentially deleterious. These two factors increase the average chain elongation rate of RNA polymerase II. An additional elongation factor, SII, rescues """"""""arrested"""""""" RNA polymerase iI that is unable to elongate RNA chains but remains template engaged. It does so by activating a newly described ribonuclease activity found in elongation complexes ranging from bacteria to humans. TFIIF, SII, and SIII can potentially control the output of many genes, yet virtually nothing is known about the spectrum of genes whose expression is dependent upon these, and other, elongation factors. We propose to define arrest sties by systematic mutagenesis and to establish an assay that measures their function in living cells. Mapping experiments will enable us to define the molecular architecture of the portions of RNA polymerase Ii that ar important for catalyzing chain elongation and harboring the nascent RNA. Experiments are planned to observe the changing relationship between the growing RNA chain and RNA polymerase Ii proposed to take place in complexes as they lose elongation competence and come to rely on elongation factor rescue. We will use an arrest-prone mutant RNA polymerase II identified in yeast, and yeast with other known mutations in the elongation machinery, to define the in vivo requirements for elongation factors and DNA sequences that precipitate a requirement for elongation factor assistance during gene expression. The cytogenetic location of the human SII gene will be identified to determine if it s a candidate gene for any known inheritable diseases. This study will provide valuable insight into the fundamental process of RNA synthesis which will improve our understanding of normal and disease states at the molecular level.
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