Severson, Mandi Clarissa2017-10-122017-10-122017https://hdl.handle.net/1794/2289049 pages. A thesis presented to the Department of Biology and the Clark Honors College of the University of Oregon in partial fulfillment of the requirements for degree of Bachelor of Science, Spring 2017RNA Polymerase II (RNAP II) is an enzyme that catalyzes the synthesis of the majority of mRNA in eukaryotic cells, making it essential for the first step of gene expression and vital for survival. The process of transcription occurs in every living cell, which makes understanding its mechansims all the more important. RNAP II consists of 12 subunits, of which Rpb1 is the largest. The Rpb1 subunit participates in RNA elongation in the active site of the enzyme. Residues located in Rpb1 have also been suggested to have a potential role in backtracking and arrest.3 Backtracking occurs when RNAP II moves backward along the DNA and the 3’ end of the RNA becomes dislodged from the active site. The backtracked RNA, which extrudes from the pore of the enzyme, binds to nearby residues creating a “backtrack site.”3 If the polymerase has not backtracked extensively, these interactions will be weak and RNAP II can spontaneously resume transcription. However, if the polymerase has backtracked extensively, forward elongation stops and RNAP II arrests.3 This study had two primary focuses: creating mutations in budding yeast (Saccharomyces cerevisiae) Rpb1 residues that interact with backtracked RNA and characterizing Rpb1 mutants via sensitivity tests involving mycophenolic acid and canavanine to determine the speed and accuracy of the mutants respectively. Creating and characterizing these mutant polymerases will give insight into what happens when the backtrack site is destabilized, which in turn could elucidate why backtrack site residues have been conserved in the first place.en-USCreative Commons BY-NC-ND 4.0-USTranscriptionRNABacktrackingMutationsEnzymeDNAThesis/Dissertation