Integrated Calendar

Transcription elongation by RNA polymerase (RNAP) is a complex process involving binding of nucleotides, conformational changes, catalytic steps, and translocation on the DNA template. The processive elongation is interspersed with transcriptional pauses, which play critical roles in coordinating transcription with processes such as translation, splicing and DNA repair. While many mechanisms contributing to pausing have been characterized, it is not clear how transcriptional dynamics of RNA polymerase change at pause sites, and how those dynamics lead to the formation of various paused states. I will present high-resolution optical tweezers experiments in which we characterize the transcription of individual E. coli RNA polymerase molecules through repeating templates. Combining the assay with novel methods of data analysis, we were able to separately investigate the pausing dynamics at different sites for pauses as short as 100 msec, and test how these dynamics are affected by applied force, backtracking and RNA structure formation. Our experiments revealed that: 1. Multiple mechanisms act in synergy to promote pausing in a site-specific manner; 2. RNA structure interacts primarily with the pretranslocated state of RNAP and can both promote or prevent pausing; 3. Backtracked pause states are formed in a site-specific manner and can only be accessed from preexisting paused states.
In the second part of the talk I will discuss the application of optical tweezers towards characterizing the stepping behavior of RNA polymerase during active elongation. Individual base-pair steps (~ 3.4 Å) have been observed before in optical tweezers assays but only anecdotally and for short segments of transcription traces. By combining an ultra-high resolution optical tweezers system with a Large-state-space Hidden Markov Model step finding algorithm, we are now able to obtain for the first time full, extended molecular trajectories of RNAP with single base-pair resolution.