Single Molecule Investigation of RNA Polymerase I using Multiplexed Tethered Particle Motion

Abstract

In eukaryotic cells, RNA Polymerase I (Pol I), which produces ribosomal RNA (rRNA), catalyzes up to 80% of all transcriptional activity in growing cells, and its regulation is an emerging field of study for anti-cancer drug development. In order to gather more comprehensive information about ribosomal RNA production by Pol I, we conducted single molecule experiments to characterize rates and pauses in the elongation phase of transcription by wild-type and mutant Pol I. Transcription by individual molecules was based on real-time measurements using custom multiplexed Tethered Particle Motion (TPM) microscopy. Microspheres were attached to one end of a DNA template engaged in an elongation complex with a Pol I molecule immmuno-imobilized on a coverslip. The range of Brownian motion of the microsphere was a function of the tether length that in turn depended on the progress of the polymerase. Either the lack of a full complement of associated factors or the constitution of in vitro transcription assay limited of polymerase activity to only 1-2%. In order to increase the throughput of the transcription assay, we increased the field of view and implemented a particle-tracking algorithm to track a high number of beads simultaneously in real time. The time courses of rare transcription events were analyzed for velocity and pausing. The processivity of Pol I without tension on the DNA template was remarkably lower than observed in studies of Pol II transcribing DNA templates under a few picoNewtons of tension. Furthermore, as reported previously based on bulk experiments, the average elongation rate of an F1205H mutant was lower than that of wild-type Pol I. At least six possible pause sites were also identified including one recently found in bulk transcription study.