The biophysics on nucleic acid based enzymes: Optical trapping studies of lambda exonuclease, RecBCD, and RNA polymerase

Nucleic acids (DNA and RNA) are central to life and so are the enzymes that interact with them. Recently, single molecule optical trapping techniques have been used to study some of these enzymes, allowing for the application of force to individual molecules. I have used single molecule optical trapping methods to study three nucleic acid based enzymes: lambda exonuclease, RecBCD helicase, and RNA polymerase (RNAP). Lambda exonuclease and RecBCD both degrade one of the two strands of DNA to leave long single stranded overhangs which are used in DNA recombination and repair. For lambda exonuclease, we detected a previously unknown sequence-dependent pausing behavior, and we used both single molecule techniques and bulk biochemical studies to determine the causative sequence for the pausing. For RecBCD, we observed spontaneous changes in enzyme velocity along with rapid reversal of the enzyme at high opposing loads, indicating that the enzyme is a complex multi-state motor.;RNAP carries out transcription---the first step in gene expression---and is therefore highly regulated. I present the first experiments in which force was applied directly to the nascent RNA during transcription. When forces >18 pN were applied to the RNA (sufficient to disrupt all RNA secondary structure), we observed that elongation and pausing kinetics of the polymerase were unaltered when compared to experiments where force was applied to the template DNA. This allowed us to rule out the possibility that RNA hairpins cause the short lifetime "ubiquitous" pauses observed in previous single molecule studies, and suggested that the RNA is held in place by direct protein-RNA contacts.