| Autor: |
Raashi, Sreenivasan, Irina A, Shkel, Munish, Chhabra, Amanda, Drennan, Sara, Heitkamp, Hao-Che, Wang, Malavika A, Sridevi, Dylan, Plaskon, Christina, McNerney, Katelyn, Callies, Clare K, Cimperman, M Thomas, Record |
| Rok vydání: |
2020 |
| Předmět: |
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| Zdroj: |
Biochemistry |
| ISSN: |
1520-4995 |
| Popis: |
FRET (fluorescence resonance energy transfer) between far-upstream(−100) and downstream(+14) cyanine dyes showed extensive bending/wrapping of λP(R) promoter DNA on E. coli RNA polymerase (RNAP) in closed and open complexes (CC, OC). Here we determine the kinetics and mechanism of DNA bending/wrapping by FRET and of formation of RNAP contacts with −100 and +14 DNA by single-dye protein-induced fluorescence enhancements (PIFE). FRET/PIFE kinetics exhibit two phases: rapidly-reversible steps forming a CC ensemble ({CC} of four intermediates (initial (RP(C)), early (I(1E)), mid- (I(1M)), late (I(1L))), followed by conversion of {CC} to OC via I(1L). FRET and PIFE are first observed for I(1E), not RP(c). FRET/PIFE together reveal large-scale bending/wrapping of upstream and downstream DNA as RP(C) advances to I(1E), reducing −100/+14 distance to ~75Å and making RNAP-DNA contacts at −100 and +14. We propose that far-upstream DNA wraps on the upper β’-clamp while downstream DNA contacts the top of the β-pincer in I(1E). Converting I(1E) to I(1M) (~1s time-scale) reduces FRET efficiency with little change in −100/+14PIFE, interpreted as clamp-opening that moves far-upstream DNA (on β’) away from downstream DNA (on β) to increase the −100/+14 distance by ~14Å. FRET increases greatly in converting I(1M) to I(1L), indicating bending of downstream duplex DNA into the clamp and clamp-closing to reduce the −100/+14 distance by ~21Å. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I(1L) converts to the initial unstable OC (I(2)). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream-binding, DNA-bending transcription activators are discussed. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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