Keynote
Investigating Nucleic Acid Synthesis at the level of Single Molecules

Single-molecule techniques have tremendously over the past few years and are now capable of in-depth reporting of enzyme dynamics. A primary advance of our in vitro techniques has been to make them high throughput. With this approach, we are able to investigate enzymatic activity on hundreds of tethered DNA or RNA molecules. Via two concrete examples, I will describe how this allows us to study different aspects of nucleic acid synthesis.

I will first describe our recent studies in which we characterize the elongation dynamics of the model RNA-dependent RNA polymerases (RdRps) from bacteriophage phi6 (P2) and from poliovirus (3Dpol), respectively, on RNA templates of kb-lengths, with or without antiviral nucleotide analogues. These studies form new approaches that may ultimately link to the development of antiviral therapeutics. Experiments on both RdRps demonstrate that their fidelity and incorporation of nucleotide analogues are governed by low fidelity, catalytically competent pauses. They also lay bare a new mechanism of action for nucleotide analogue drugs, by showing, first, that T-1106 presents a strong antiviral activity on polio virus-infected cells without the cellular toxicity of ribavirin, and second that its incorporation of T-1106 opposite an adenosine results in pauses originating in polymerase backtracks. These findings demonstrate the potential of high-throughput single-molecule techniques for drug characterization.

I will next describe our ongoing studies of the kinetics during transcriptional elongation by E. coli RNA polymerase (RNAp). We subject the RNAp to a wide variety of different conditions (NTP concentration, force, nucleotide analogues, etc.) and find that we are able to fit its response to a simple model. By examining in particular the tendency of this polymerase to pause on very long timescales, we shed new light on the phenomenon of polymerase backtracking.

Jointly, these two studies illustrate how high-throughput single-molecule studies allow us to probe the mechanisms that underlie nucleic acids synthesis in greater depth than was previously possible.