Preventing Transcriptional Conflict – PrTxConf

Gene transcription is a tightly regulated process, which is strongly licensed at initiation, extremely processive throughout elongation, and then essentially abruptly stopped at termination. Broadly speaking, transcription termination stops RNA polymerase (RNAP) in its tracks, and from this arrested state both RNA and RNAP are released from the DNA. If elongating RNAP is not properly terminated, its stability on DNA ensures that it may continue transcribing over large distances, potentially interfering with other genomic processes, which take place on these downstream DNA sequences. Thus for instance in humans the mis-termination of cryptic antisense transcription by dysfunctional senataxin, an RNA translocase, leads to developmental defects. Despite recent advances, the structural and kinetic analysis of paused, stalled, and terminating RNAP has remained particularly challenging, because these states and their intermediates are typically transient and difficult to synchronize.

In this project we will use cutting-edge single-molecule approaches including electron cryo-microscopy (cryo-EM) and correlative single-molecule nanomanipulation and fluorescence to address mechanistic questions pertaining to transcription termination. These complementary approaches will provide insight into both kinetic and structural aspects of paused and terminating RNAP. These include the role of conformational changes in RNAP during transcriptional pausing and termination, and in particular opening and closing of the "crab-claw" structure of RNAP about DNA. We will also analyze the role of transcription factors, which rescue backtracked RNA polymerase: their binding to RNAP is seen to be dynamic in single molecule experiments. However, we lack a complete mechanistic and structural understanding of the observed states. What structural states do these complexes assume as seen by cryo-EM analysis? Finally, this project will also focus on extrinsic termination processes, which are involved in regulating cryptic and antisense termination. These latter processes involve the interactions between helicases and polymerases, underscoring their dynamic nature and the requirement for real-time methods such as single-molecule nanomanipulation and fluorescence to understand their interactions.