T7 RNA polymerase elongation complex structure and movement

Jianbin Huang, Rui J Sousa

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

We have characterized T7RNAP elongation complexes (ECs) halted at different positions on a single template using a combination of digestion with exonuclease III, λ exonuclease, RNAse T1, and treatment with KMnO4. Our results indicate that the transcription bubble is approximately nine bases long and that the RNA:DNA hybrid is 7-8 bp in size. An additional four to six bases of RNA immediately 5' to the hybrid interact with the RNAP, probably with a site on the N-terminal domain. When ECs with transcripts of different length were probed in the presence or absence of the incoming NTP we found that the position of the EC on the template and the RNA shifted downstream upon NTP binding. NTP binding also restricted the lateral mobility of the complex on the template. Our results indicate that, in the absence of bound NTP, the RNAP is relatively free to slide on the template around a position that usually lies one to two bases upstream of the position from which NTP binding and bond formation occur. NTP binding stabilizes the RNAP in the post-translocated position and keeps it from sliding upstream, either due directly to RNAP:NTP:template interactions, or to an isomerization which causes the fingers subdomain of the RNAP to clamp down on the downstream end of the template strand. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)347-358
Number of pages12
JournalJournal of Molecular Biology
Volume303
Issue number3
DOIs
StatePublished - Oct 27 2000

Fingerprint

RNA
Exonucleases
Fingers
Digestion
DNA
bacteriophage T7 RNA polymerase
exodeoxyribonuclease III

Keywords

  • Elongation
  • Motor proteins
  • RNA polymerase
  • Transcription
  • Translocation

ASJC Scopus subject areas

  • Virology

Cite this

T7 RNA polymerase elongation complex structure and movement. / Huang, Jianbin; Sousa, Rui J.

In: Journal of Molecular Biology, Vol. 303, No. 3, 27.10.2000, p. 347-358.

Research output: Contribution to journalArticle

@article{5adbb70e0f4c4a78b595a06c47ce2014,
title = "T7 RNA polymerase elongation complex structure and movement",
abstract = "We have characterized T7RNAP elongation complexes (ECs) halted at different positions on a single template using a combination of digestion with exonuclease III, λ exonuclease, RNAse T1, and treatment with KMnO4. Our results indicate that the transcription bubble is approximately nine bases long and that the RNA:DNA hybrid is 7-8 bp in size. An additional four to six bases of RNA immediately 5' to the hybrid interact with the RNAP, probably with a site on the N-terminal domain. When ECs with transcripts of different length were probed in the presence or absence of the incoming NTP we found that the position of the EC on the template and the RNA shifted downstream upon NTP binding. NTP binding also restricted the lateral mobility of the complex on the template. Our results indicate that, in the absence of bound NTP, the RNAP is relatively free to slide on the template around a position that usually lies one to two bases upstream of the position from which NTP binding and bond formation occur. NTP binding stabilizes the RNAP in the post-translocated position and keeps it from sliding upstream, either due directly to RNAP:NTP:template interactions, or to an isomerization which causes the fingers subdomain of the RNAP to clamp down on the downstream end of the template strand. (C) 2000 Academic Press.",
keywords = "Elongation, Motor proteins, RNA polymerase, Transcription, Translocation",
author = "Jianbin Huang and Sousa, {Rui J}",
year = "2000",
month = "10",
day = "27",
doi = "10.1006/jmbi.2000.4150",
language = "English (US)",
volume = "303",
pages = "347--358",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "3",

}

TY - JOUR

T1 - T7 RNA polymerase elongation complex structure and movement

AU - Huang, Jianbin

AU - Sousa, Rui J

PY - 2000/10/27

Y1 - 2000/10/27

N2 - We have characterized T7RNAP elongation complexes (ECs) halted at different positions on a single template using a combination of digestion with exonuclease III, λ exonuclease, RNAse T1, and treatment with KMnO4. Our results indicate that the transcription bubble is approximately nine bases long and that the RNA:DNA hybrid is 7-8 bp in size. An additional four to six bases of RNA immediately 5' to the hybrid interact with the RNAP, probably with a site on the N-terminal domain. When ECs with transcripts of different length were probed in the presence or absence of the incoming NTP we found that the position of the EC on the template and the RNA shifted downstream upon NTP binding. NTP binding also restricted the lateral mobility of the complex on the template. Our results indicate that, in the absence of bound NTP, the RNAP is relatively free to slide on the template around a position that usually lies one to two bases upstream of the position from which NTP binding and bond formation occur. NTP binding stabilizes the RNAP in the post-translocated position and keeps it from sliding upstream, either due directly to RNAP:NTP:template interactions, or to an isomerization which causes the fingers subdomain of the RNAP to clamp down on the downstream end of the template strand. (C) 2000 Academic Press.

AB - We have characterized T7RNAP elongation complexes (ECs) halted at different positions on a single template using a combination of digestion with exonuclease III, λ exonuclease, RNAse T1, and treatment with KMnO4. Our results indicate that the transcription bubble is approximately nine bases long and that the RNA:DNA hybrid is 7-8 bp in size. An additional four to six bases of RNA immediately 5' to the hybrid interact with the RNAP, probably with a site on the N-terminal domain. When ECs with transcripts of different length were probed in the presence or absence of the incoming NTP we found that the position of the EC on the template and the RNA shifted downstream upon NTP binding. NTP binding also restricted the lateral mobility of the complex on the template. Our results indicate that, in the absence of bound NTP, the RNAP is relatively free to slide on the template around a position that usually lies one to two bases upstream of the position from which NTP binding and bond formation occur. NTP binding stabilizes the RNAP in the post-translocated position and keeps it from sliding upstream, either due directly to RNAP:NTP:template interactions, or to an isomerization which causes the fingers subdomain of the RNAP to clamp down on the downstream end of the template strand. (C) 2000 Academic Press.

KW - Elongation

KW - Motor proteins

KW - RNA polymerase

KW - Transcription

KW - Translocation

UR - http://www.scopus.com/inward/record.url?scp=0034721946&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034721946&partnerID=8YFLogxK

U2 - 10.1006/jmbi.2000.4150

DO - 10.1006/jmbi.2000.4150

M3 - Article

C2 - 11031112

AN - SCOPUS:0034721946

VL - 303

SP - 347

EP - 358

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 3

ER -