Abstract
Lac repressor is a transcription factor that represses expression of genes in the lac operon. The mechanism by which Lac repressor regulates transcription initiation remains unclear, but it is understood that in the presence of Lac repressor, open complex formation is inhibited. In this thesis, we fluorescently label a single-cysteine mutant of the Lac repressor that specifically binds to O1 operator and responds to inducer IPTG in bulk and single molecule in vitro experiments. The labeling efficiency is 26% of monomers, corresponding to 70% of all tetramers in solution having at least one cysteine labeled. The labeled construct, dubbed TAMRA-Lac, was found to be 41% active using an Electrophoretic Mobility Shift Assay. On the single molecule level, the labeled construct binds 57% of operator DNA versus 0.86% of nonoperator DNA and 3.8% of operator DNA after IPTG induction. I used this active and labeled construct in an attempt to probe the mechanism by which Lac repressor regulates transcription initiation on the single molecule level.