Closing and opening of the RNA polymerase trigger loop

Abstract

<jats:p>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.</jats:p>

The Rate-limiting Step of DNA Synthesis by DNA Polymerase Occurs in the Fingers-closed Conformation

“…This characteristic shape of distributions points to the presence of several sequential rate-limiting steps, as expected from the presence of multiple cycles of nucleotide addition for a single KF-binding event. 33 , 34 , 35 There was not a large shift in the polymerisation times between 5-nt and 10-nt extensions, with the mean polymerisation time only increasing from 1.9 s for 5-nt extensions to 2.2 s for 10-nt extensions. This suggested that the bases between the 5–10 positions may be incorporated faster that the bases between positions 1–5.…”

Structural and mechanistic basis of reiterative transcription initiation

“…In the crystal structures of this work, as in the crystal structures of refs. 27 and 28 , RNA products generated by reiterative transcription initiation were limited in length because further RNA extension was blocked by the presence of the σ finger in the RNAP active-center cleft and by crystal-lattice constraints that prevented displacement of the σ finger from the RNAP active-center cleft ( 35 , 37 ), opening of the RNAP clamp ( 39 – 41 ), or any other conformational change that could open a path for further extension of RNA and for extrusion of RNA from the RNAP active-center cleft. One hypothesis is that, in solution, complete displacement of the σ finger from the RNAP active-center cleft channel could allow long RNA products generated in reiterative transcription initiation to exit the RNAP active-center cleft through the RNAP RNA-exit channel, the same exit route used by RNA in standard transcription ( 28 ).…”

Probing steps in DNA transcription using single-molecule methods

“…Previous studies using smFRET have revealed various aspects of dynamics during the transcription initiation and transition states ( 17 , 26 , 31 , 62 , 69 , 70 , 107 , 108 , 109 , 110 , 111 ). Typically, to visualize the conformational changes during initiation, FRET-pair dyes, such as Cy3 and Cy5, can be attached to RNAP subunits ( Fig.…”