Oral Presentation 16th Asian Conference on Transcription 2019

Transcription reinitiation by E. coli RNA polymerase recycling and diffusing on DNA after intrinsic termination (1096)

Wooyoung Kang 1 , Kook Sun Ha 2 , Heesoo Uhm 1 , Kyuhyong Park 1 , Ja Yil Lee 3 , Sungchul Hohng 1 , Changwon Kang 2
  1. Department of Physics and Astronomy, Seoul National University, Seoul, Republic of Korea
  2. Korea Advanced Institute of Science and Technology, Deajeon, Republic of Korea
  3. School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea

It is unknown how transcription termination complexes are disassembled, especially in what order the essential components dissociate from transcription complexes. We primarily used single-molecule fluorescence measurements to examine the dissociations of fluorescently labeled Cy3-RNA transcript and Cy5-DNA template from unlabeled RNA polymerase (RNAP) in E. coli intrinsic termination, and their post-terminational fates. RNA product release precedes RNAP dissociation from immobilized DNA template much more often than their concurrent dissociations in intrinsic termination. As termination is defined by the release of product RNA from transcription complex, the subsequent retention of RNAP on DNA template constitutes a previously unidentified stage, termed as 'recycling.' In experiments with Cy5 placed on RpoD (σ70) factor instead of DNA, Cy5-σ never dissociates before Cy3-RNA. Post-terminational retention of RNAP on DNA is little affected by the presence of transcription factor σ70, NusA or NusG. RNAP's post-terminational possession of σ and diffusion on DNA during the recycling stage allow for transcription 'reinitiation,' which occurs not only on a downstream promoter oriented in the sense direction but also on the original promoter. Thus, RNAP can diffuse downward and upward on DNA template for reinitiation on any promoter that can be reached within a diffusion lifetime. With flipping of RNAP on DNA, antisense reinitiation at an oppositely oriented promoter could be possible. Furthermore, σ supplement increases the reinitiation efficiency, suggesting that even core enzyme can become reinitiation-competent during the recycling stage. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on DNA template for reinitiation most times.