RNA Polymerase II Translocation and Fidelity are Governed by Trigger Loop Dynamics: A Single-Molecule Study

AbstractThe carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

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Conserved Trigger Loop Histidine of RNA Polymerase II Functions as a Positional Catalyst Primarily through Steric Effects

Biochemistry ◽

10.1021/acs.biochem.1c00528 ◽

2021 ◽

Author(s):

Michael Z. Palo ◽

Junqiao Zhu ◽

Tatiana V. Mishanina ◽

Robert Landick

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Steric Effects ◽

Trigger Loop

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Closing and opening of the RNA polymerase trigger loop

10.1101/817080 ◽

2019 ◽

Cited By ~ 1

Author(s):

Abhishek Mazumder ◽

Miaoxin Lin ◽

Achillefs N. Kapanidis ◽

Richard H. Ebright

Keyword(s):

Rna Polymerase ◽

Fluorescent Probe ◽

Active Center ◽

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Single Molecule Fluorescence ◽

Structural Element ◽

Trigger Loop ◽

Nucleotide Addition

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.

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Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1421067112 ◽

2014 ◽

Vol 112 (3) ◽

pp. 743-748 ◽

Cited By ~ 31

Author(s):

Yara X. Mejia ◽

Evgeny Nudler ◽

Carlos Bustamante

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Single Molecule ◽

Conformational Changes ◽

Elongation Rate ◽

Nucleoside Triphosphate ◽

Transcription Rate ◽

Nucleotide Analogs ◽

Catalytic Center ◽

Trigger Loop

Two components of the RNA polymerase (RNAP) catalytic center, the bridge helix and the trigger loop (TL), have been linked with changes in elongation rate and pausing. Here, single molecule experiments with the WT and two TL-tip mutants of the Escherichia coli enzyme reveal that tip mutations modulate RNAP’s pause-free velocity, identifying TL conformational changes as one of two rate-determining steps in elongation. Consistent with this observation, we find a direct correlation between helix propensity of the modified amino acid and pause-free velocity. Moreover, nucleotide analogs affect transcription rate, suggesting that their binding energy also influences TL folding. A kinetic model in which elongation occurs in two steps, TL folding on nucleoside triphosphate (NTP) binding followed by NTP incorporation/pyrophosphate release, quantitatively accounts for these results. The TL plays no role in pause recovery remaining unfolded during a pause. This model suggests a finely tuned mechanism that balances transcription speed and fidelity.

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