[대학원 생명과학과 세미나 안내]
연사 : 강진영 교수 (KAIST 화학과)
연제 : Structural study on prokaryotic transcription - how RNA polymerases pause and go
일시 : 2019년 5월 10일 (금) 오후 5시
장소 : 하나과학관 A동 B131호
초청교수 : 우재성 교수
Abstract
The transcriptional pausing plays pivotal roles in transcription initiation, elongation, termination, RNA folding, translation, and genome stability. RNA polymerase (RNAP) pauses at every ~100 base pairs during transcription in the model bacterium, E. coli. In bacteria, secondary structure of nascent RNA can increase pause lifetimes ten-fold or more. Despite its central life-maintaining function, the underlying molecular mechanism of transcriptional pausing has remained elusive. To probe this, we determined a 3.8 Å cryo-electron microscopy (cryo-EM) structure of an E. coli his pause elongation complex (hisPEC), which is stabilized by a nascent RNA hairpin derived from the leader sequence of the histidine synthesis operon. We found that i) the double-stranded A-form RNA hairpin stem forms within the RNA exit channel of RNAP, ii) the DNA:RNA hybrid is trapped in a distinctive half-translocated conformation, and iii) mobile domains of the RNAP (dock, clamp, jaw, shelf and SI3) rearrange in a concerted manner to maintain the RNA hairpin-stabilized paused state. Although the transcriptional pause is dynamically controlled by transcription factors for the gene expression regulation, how the factors can adjust the pausing at the atomic level remains unknown. To address this question, we focused on the two prominent anti-pausing proteins, NusG and RfaH, which belong to the only conserved transcription factor family in all domains of life. Cryo-EM structures of elongation complexes bound with NusG and RfaH, at 3.7 Å and 3.5 Å resolution, respectively, revealed that NusG and RfaH bind to the β protrusion, β gate loop, and β’ clamp helices of RNAP, stabilizing the RNAP in a conformation of active elongation state. While NusG is a ubiquitous pausing inhibitor, RfaH associates with an RNAP in a sequence-specific manner. In the RfaH-elongation complex structure, RfaH interacts with ops (operon polarity repressor) sequence forming a short hairpin on the non-template DNA. While both NusG and RfaH reduce backtrack pausing, which is transient transcription stalling by RNAP’s backward movement, only RfaH blocks RNA hairpin-stabilized pausing by binding to the elongation complex tightly enough to resist RNAP swiveling. Based on our results, we propose that the pause RNA hairpin stabilizes global conformational changes in the RNAP that secures the paused state and NusG and RfaH enhance transcription elongation by maintaining the active conformation of the elongation complex.