Determining the Rate of Transcription of T7 RNA Polymerase Using Single Molecule Fluorescence Imaging

It is important to understand the many factors impacting the rate at which an RNA polymerase incorporates nucleotides. The transcription rate of T7 RNA polymerase has been determined using single molecule fluorescence microscopy. A Cy3 labeled circular 45nt ssDNA molecule was used to monitor the transcription process. T7 RNA polymerase was used because it is a single subunit polymerase that does not need any cofactors and will transcribe single-stranded DNA circles that do not contain a promoter. The transcription was monitored by measuring the quasi-periodic change in intensity associated with the transit of the probe through the polymerase as the DNA is transcribed. The time between these intensity changes of the Cy3 molecule represents the time it takes the polymerase to transcribe the circle once. Transcription rates were determined at a variety of NTP concentrations. Because glass can affect how the enzyme works, the surface of the glass was coated with poly-L-lysine in some of the experiments. The poly-L-lysine was used to keep the T7 RNAP from touching the glass surface. In order to extend the observation time, factors affecting the photostability of the Cy3 probe were evaluated using determinations of the photochemical half-life