Macromolecular crowding alters transcription: real-time measurements with SYBR Green II

Abstract Various factors affect transcription, including regulatory proteins, promoter sequences, ionic conditions, and nucleotide concentrations. Gel electrophoresis of radiolabeled RNA is a widely used assay to quantify transcript production in vitro. However, the use of radioactive reagents requires hazardous chemical training as well as specialized protocols and imaging systems. Moreover, dynamic monitoring of in vitro transcription could provide insight into conditions affecting the reaction. Widely available microplate readers and quantitative PCR instruments could be broadly adopted to study the kinetics and yields of transcription reactions with a suitable fluorescence assay. The SYBR Green II fluorophore has high affinity for RNA and can be used to monitor RNA production from in vitro transcription assays. In experiments described herein, the effect of macromolecular crowding agents on transcription by Escherichia coli RNA polymerase was characterized. Polyethylene glycol (PEG) 2000 inhibited transcription more than PEG 8000 which displayed re-entrant behavior as a function of concentration. Glycerol had little effect and Dextran 70 stimulated transcription by approximately 20%.