Abstract
The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an "unfolded"/"open" state that allows an NTP substrate to enter the active center and a "folded"/"closed" state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription elongation.
Highlights
The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an “unfolded”/“open” state that allows an NTP substrate to enter the active center and a “folded”/“closed” state that holds the NTP substrate in the active center
Crystal structures of transcription elongation complexes indicate that the TL can adopt 1) an “unfolded,” or “open,” TL conformation that allows an NTP to enter the RNAP active center; and 2) a “folded,” or “closed,” TL conformation that holds an NTP in the RNAP active center
Using single-molecule fluorescence spectroscopy to monitor distances between a probe incorporated into the TL and a probe incorporated elsewhere in the transcription elongation complex, we show that TL closing and opening occur in solution, define time scales and functional roles of TL closing and opening, and, most crucially, demonstrate that one cycle of TL closing and opening occurs in each nucleotide-addition cycle
Summary
It further has been hypothesized that the TL returns to its initial, unfolded, open conformational state on or after phosphodiester-bond formation, thereby reopening the RNAP active center and permitting pyrophosphate release and RNAP translocation [2, 5, 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28] According to these proposals, an RNAP active-center conformational cycle, comprising TL closing followed by TL opening, is coupled to each nucleotide-addition cycle [2, 5, 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28].
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