Professor Laura Finzi, Department of Physics, Emory University of Arts and Sciences, Georgia, USA

Single-Molecule Investigation of Transcriptional Regulation

I will describe how single-molecule techniques can be used to understand the molecular mechanisms that regulate transcription of genes and characterize the kinetics of transcriptional elongation by RNA polymerases. In the first part of my talk, I will describe the use of repressors that regulate the lysogeny-to-lysis genetic switch in the lambda and the 186 bacteriophages as model systems. Although lambda is a paradigmatic transcriptional repressor and one might guess that there is little left to understand about the mechanism, using magnetic tweezers, tethered particle and atomic force microscopy, we characterized how specific and non-specific binding of repressor protein to DNA as well as DNA negative supercoiling make the lambda switch both robust and sensitive. Distinct affinities for different DNA sequences are also important to the regulatory function of the disc-shaped 186 bacteriophage repressor which recapitulates features of various transcription factors. We have visualized the ensemble of wrapped and looped DNA configurations that are thought to modulate repression and are now assaying transcription dynamics.

Following that, I will describe our direct characterization of transcriptional elongation by RNA polymerase I (Pol I) at the single molecule level. RNA 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 the kinetics of elongation by Pol I is lacking. Tethered particle microscopy experiments have shown that the average rate of Pol I elongation is 20 nt/s, but the maximum rate is approximately 50 nt/s, comparable to that estimated in vivo. Furthermore, addition of RNA endonucleases to the elongation experiments enhanced processivity and shortened pauses. These data are consistent with the model that high RNA Pol I density on rDNA results in enhanced transcription elongation kinetics by minimizing pausing by Pol I and interference from R-loop formation.