A Single Molecule Perspective of Elongation by RNA Polymerase I

Abstract

RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of total transcription in a growing cell. Despite its central role in transcription and fundamental role in cell growth and proliferation, a detailed understanding of kinetics of transcription by Pol I is lacking. We present the first direct characterization of Pol I transcription elongation at the single molecule level. Tethered particle microscopy shows that the pause-free rate measured for Pol I, approximately 50 nt/s, is comparable to the in vivo rate estimated from the number of active genes, the cell division rate and the number of engaged polymerases determined from EM images. Furthermore, comparison of elongation traces in the presence or absence of RNAse A & T1, reveals strong interactions between the nascent RNA and the DNA tether that might modulate transcription elongation in vivo. In the presence of RNAse, Pol I processivity is higher than in the absence of RNAse. Furthermore, when the nascent transcript is not digested by RNAse, large tether length fluctuations are noticed which yield an apparently slower average elongation rate of approximately 20 nt/s.