Fluorescence Assay for Polymerase Arrival Rates

Abstract

To engineer complex synthetic biological systems will require modulardesign, assembly, and characterization strategies. The RNApolymerase arrival rate (PAR) is defined to be the rate that RNApolymerases arrive at a specified location on the DNA. Designing andcharacterizing biological modules in terms of RNA polymerase arrivalrates provides for many advantages in the construction and modeling ofbiological systems.PARMESAN is an in vitro method for measuring polymerase arrival ratesusing pyrrolo-dC, a fluorescent DNA base that can substitute forcytosine. Pyrrolo-dC shows a detectable fluorescence difference whenin single-stranded versus double-stranded DNA. During transcription,RNA polymerase separates the two strands of DNA, leading to a changein the fluorescence of pyrrolo-dC. By incorporating pyrrolo-dC atspecific locations in the DNA, fluorescence changes can be taken as adirect measurement of the polymerase arrival rate.