Translocation by T7 RNA polymerase: A sensitively poised brownian ratchet

Qing Guo, Rui J Sousa

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

Studies of halted T7 RNA polymerase (T7RNAP) elongation complexes (ECs) or of T7RNAP transcription against roadblocks due to DNA-bound proteins indicate that T7RNAP translocates via a passive Brownian ratchet mechanism. Crystal structures of T7RNAP ECs suggest that translocation involves an active power-stroke. However, neither solution studies of halted or slowed T7RNAP ECs, nor crystal structures of static complexes, are necessarily relevant to how T7RNAP translocates during rapid elongation. A recent single molecule study of actively elongating T7RNAPs provides support for the Brownian ratchet mechanism. Here, we obtain additional evidence for the existence of a Brownian ratchet during active T7RNAP elongation by showing that both rapidly elongating and halted complexes are equally sensitive to pyrophosphate. Using chemical nucleases tethered to the polymerase we achieve sub-ångström resolution in measuring the average position of halted T7RNAP ECs and find that the positional equilibrium of the EC is sensitively poised between pre-translocated and post-translocated states. This may be important in maximizing the sensitivity of the polymerase to sequences that cause pausing or termination. We also confirm that a crystallographically observed disorder to order transition in a loop formed by residues 589-612 also occurs in solution and is coupled to pyrophosphate or NTP release. This transition allows the loop to make interactions with the DNA that help stabilize the laterally mobile, ligand-free EC against dissociation.

Original languageEnglish (US)
Pages (from-to)241-254
Number of pages14
JournalJournal of Molecular Biology
Volume358
Issue number1
DOIs
StatePublished - Apr 21 2006

Fingerprint

RNA Polymerase I
bacteriophage T7 RNA polymerase
DNA
Stroke
Ligands

Keywords

  • Brownian ratchet
  • T7 RNA polymerase
  • Transcription dynamics
  • Transcription elongation
  • Translocation

ASJC Scopus subject areas

  • Virology

Cite this

Translocation by T7 RNA polymerase : A sensitively poised brownian ratchet. / Guo, Qing; Sousa, Rui J.

In: Journal of Molecular Biology, Vol. 358, No. 1, 21.04.2006, p. 241-254.

Research output: Contribution to journalArticle

@article{0497dcc534b4413299cd96f407f4913f,
title = "Translocation by T7 RNA polymerase: A sensitively poised brownian ratchet",
abstract = "Studies of halted T7 RNA polymerase (T7RNAP) elongation complexes (ECs) or of T7RNAP transcription against roadblocks due to DNA-bound proteins indicate that T7RNAP translocates via a passive Brownian ratchet mechanism. Crystal structures of T7RNAP ECs suggest that translocation involves an active power-stroke. However, neither solution studies of halted or slowed T7RNAP ECs, nor crystal structures of static complexes, are necessarily relevant to how T7RNAP translocates during rapid elongation. A recent single molecule study of actively elongating T7RNAPs provides support for the Brownian ratchet mechanism. Here, we obtain additional evidence for the existence of a Brownian ratchet during active T7RNAP elongation by showing that both rapidly elongating and halted complexes are equally sensitive to pyrophosphate. Using chemical nucleases tethered to the polymerase we achieve sub-{\aa}ngstr{\"o}m resolution in measuring the average position of halted T7RNAP ECs and find that the positional equilibrium of the EC is sensitively poised between pre-translocated and post-translocated states. This may be important in maximizing the sensitivity of the polymerase to sequences that cause pausing or termination. We also confirm that a crystallographically observed disorder to order transition in a loop formed by residues 589-612 also occurs in solution and is coupled to pyrophosphate or NTP release. This transition allows the loop to make interactions with the DNA that help stabilize the laterally mobile, ligand-free EC against dissociation.",
keywords = "Brownian ratchet, T7 RNA polymerase, Transcription dynamics, Transcription elongation, Translocation",
author = "Qing Guo and Sousa, {Rui J}",
year = "2006",
month = "4",
day = "21",
doi = "10.1016/j.jmb.2006.02.001",
language = "English (US)",
volume = "358",
pages = "241--254",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Translocation by T7 RNA polymerase

T2 - A sensitively poised brownian ratchet

AU - Guo, Qing

AU - Sousa, Rui J

PY - 2006/4/21

Y1 - 2006/4/21

N2 - Studies of halted T7 RNA polymerase (T7RNAP) elongation complexes (ECs) or of T7RNAP transcription against roadblocks due to DNA-bound proteins indicate that T7RNAP translocates via a passive Brownian ratchet mechanism. Crystal structures of T7RNAP ECs suggest that translocation involves an active power-stroke. However, neither solution studies of halted or slowed T7RNAP ECs, nor crystal structures of static complexes, are necessarily relevant to how T7RNAP translocates during rapid elongation. A recent single molecule study of actively elongating T7RNAPs provides support for the Brownian ratchet mechanism. Here, we obtain additional evidence for the existence of a Brownian ratchet during active T7RNAP elongation by showing that both rapidly elongating and halted complexes are equally sensitive to pyrophosphate. Using chemical nucleases tethered to the polymerase we achieve sub-ångström resolution in measuring the average position of halted T7RNAP ECs and find that the positional equilibrium of the EC is sensitively poised between pre-translocated and post-translocated states. This may be important in maximizing the sensitivity of the polymerase to sequences that cause pausing or termination. We also confirm that a crystallographically observed disorder to order transition in a loop formed by residues 589-612 also occurs in solution and is coupled to pyrophosphate or NTP release. This transition allows the loop to make interactions with the DNA that help stabilize the laterally mobile, ligand-free EC against dissociation.

AB - Studies of halted T7 RNA polymerase (T7RNAP) elongation complexes (ECs) or of T7RNAP transcription against roadblocks due to DNA-bound proteins indicate that T7RNAP translocates via a passive Brownian ratchet mechanism. Crystal structures of T7RNAP ECs suggest that translocation involves an active power-stroke. However, neither solution studies of halted or slowed T7RNAP ECs, nor crystal structures of static complexes, are necessarily relevant to how T7RNAP translocates during rapid elongation. A recent single molecule study of actively elongating T7RNAPs provides support for the Brownian ratchet mechanism. Here, we obtain additional evidence for the existence of a Brownian ratchet during active T7RNAP elongation by showing that both rapidly elongating and halted complexes are equally sensitive to pyrophosphate. Using chemical nucleases tethered to the polymerase we achieve sub-ångström resolution in measuring the average position of halted T7RNAP ECs and find that the positional equilibrium of the EC is sensitively poised between pre-translocated and post-translocated states. This may be important in maximizing the sensitivity of the polymerase to sequences that cause pausing or termination. We also confirm that a crystallographically observed disorder to order transition in a loop formed by residues 589-612 also occurs in solution and is coupled to pyrophosphate or NTP release. This transition allows the loop to make interactions with the DNA that help stabilize the laterally mobile, ligand-free EC against dissociation.

KW - Brownian ratchet

KW - T7 RNA polymerase

KW - Transcription dynamics

KW - Transcription elongation

KW - Translocation

UR - http://www.scopus.com/inward/record.url?scp=33645097171&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33645097171&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2006.02.001

DO - 10.1016/j.jmb.2006.02.001

M3 - Article

C2 - 16516229

AN - SCOPUS:33645097171

VL - 358

SP - 241

EP - 254

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 1

ER -