Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex

Kay M. Parkhurst, Robyn M. Richards, Michael Brenowitz, Lawrence J Parkhurst

Research output: Contribution to journalArticle

70 Citations (Scopus)

Abstract

Binding of the TATA-binding protein (TBP) to the 'TATA' sequences present in the promoters of eukaryotic class II genes is the first step in the sequential assembly of transcription pre-initiation complexes. Myriad structural changes, including severe bending of the DNA, accompany TBP-TATA complex formation. A detailed kinetic study has been conducted to elucidate the mechanistic details of TBP binding and DNA bending. The binding of Saccharomyces cerevisiae TBP to the adenovirus major late promoter (AdMLP) was followed in real-time through a range of temperatures and TBP concentrations using fluorescence resonance energy transfer (FRET) and stopped-flow mixing. The results of association and relaxation kinetics and equilibrium binding experiments were analyzed globally to obtain the complete kinetic and energetic profile of the reaction. This analysis reveals a complex mechanism with two intermediate species, with the DNA in the intermediates apparently bent similarly to the DNA in the final complex. TBP binding and DNA bending occur simultaneously through the multiple steps of the reaction. The first and third steps in this sequential process show nearly identical large increases in both enthalpy and entropy, whereas the middle step is highly exothermic and proceeds with a large decrease in entropy. The first intermediate is significantly populated at equilibrium and resembles the final complex both structurally and energetically. It is postulated that both this intermediate and the final complex bind transcription factor IIB in the second step of pol II pre-initiation complex assembly. A consequence of such a reactive intermediate is that the rate of assembly of transcriptionally competent pre-initiation complexes from bi-directionally bound TBP is greatly increased.

Original languageEnglish (US)
Pages (from-to)1327-1341
Number of pages15
JournalJournal of Molecular Biology
Volume289
Issue number5
DOIs
StatePublished - Jun 25 1999

Fingerprint

TATA-Box Binding Protein
DNA
Entropy
Protein Binding
Transcription Factor TFIIB
Saccharomyces cerevisiae Proteins
MHC Class II Genes
Fluorescence Resonance Energy Transfer
Adenoviridae
Temperature

Keywords

  • DNA-protein interaction
  • Fluorescence resonance energy transfer (FRET)
  • TATAa
  • TBP
  • Transcription

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex. / Parkhurst, Kay M.; Richards, Robyn M.; Brenowitz, Michael; Parkhurst, Lawrence J.

In: Journal of Molecular Biology, Vol. 289, No. 5, 25.06.1999, p. 1327-1341.

Research output: Contribution to journalArticle

Parkhurst, Kay M. ; Richards, Robyn M. ; Brenowitz, Michael ; Parkhurst, Lawrence J. / Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex. In: Journal of Molecular Biology. 1999 ; Vol. 289, No. 5. pp. 1327-1341.
@article{f7da05620bb54defaad0521e186d2a9d,
title = "Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex",
abstract = "Binding of the TATA-binding protein (TBP) to the 'TATA' sequences present in the promoters of eukaryotic class II genes is the first step in the sequential assembly of transcription pre-initiation complexes. Myriad structural changes, including severe bending of the DNA, accompany TBP-TATA complex formation. A detailed kinetic study has been conducted to elucidate the mechanistic details of TBP binding and DNA bending. The binding of Saccharomyces cerevisiae TBP to the adenovirus major late promoter (AdMLP) was followed in real-time through a range of temperatures and TBP concentrations using fluorescence resonance energy transfer (FRET) and stopped-flow mixing. The results of association and relaxation kinetics and equilibrium binding experiments were analyzed globally to obtain the complete kinetic and energetic profile of the reaction. This analysis reveals a complex mechanism with two intermediate species, with the DNA in the intermediates apparently bent similarly to the DNA in the final complex. TBP binding and DNA bending occur simultaneously through the multiple steps of the reaction. The first and third steps in this sequential process show nearly identical large increases in both enthalpy and entropy, whereas the middle step is highly exothermic and proceeds with a large decrease in entropy. The first intermediate is significantly populated at equilibrium and resembles the final complex both structurally and energetically. It is postulated that both this intermediate and the final complex bind transcription factor IIB in the second step of pol II pre-initiation complex assembly. A consequence of such a reactive intermediate is that the rate of assembly of transcriptionally competent pre-initiation complexes from bi-directionally bound TBP is greatly increased.",
keywords = "DNA-protein interaction, Fluorescence resonance energy transfer (FRET), TATAa, TBP, Transcription",
author = "Parkhurst, {Kay M.} and Richards, {Robyn M.} and Michael Brenowitz and Parkhurst, {Lawrence J}",
year = "1999",
month = "6",
day = "25",
doi = "10.1006/jmbi.1999.2835",
language = "English (US)",
volume = "289",
pages = "1327--1341",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "5",

}

TY - JOUR

T1 - Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex

AU - Parkhurst, Kay M.

AU - Richards, Robyn M.

AU - Brenowitz, Michael

AU - Parkhurst, Lawrence J

PY - 1999/6/25

Y1 - 1999/6/25

N2 - Binding of the TATA-binding protein (TBP) to the 'TATA' sequences present in the promoters of eukaryotic class II genes is the first step in the sequential assembly of transcription pre-initiation complexes. Myriad structural changes, including severe bending of the DNA, accompany TBP-TATA complex formation. A detailed kinetic study has been conducted to elucidate the mechanistic details of TBP binding and DNA bending. The binding of Saccharomyces cerevisiae TBP to the adenovirus major late promoter (AdMLP) was followed in real-time through a range of temperatures and TBP concentrations using fluorescence resonance energy transfer (FRET) and stopped-flow mixing. The results of association and relaxation kinetics and equilibrium binding experiments were analyzed globally to obtain the complete kinetic and energetic profile of the reaction. This analysis reveals a complex mechanism with two intermediate species, with the DNA in the intermediates apparently bent similarly to the DNA in the final complex. TBP binding and DNA bending occur simultaneously through the multiple steps of the reaction. The first and third steps in this sequential process show nearly identical large increases in both enthalpy and entropy, whereas the middle step is highly exothermic and proceeds with a large decrease in entropy. The first intermediate is significantly populated at equilibrium and resembles the final complex both structurally and energetically. It is postulated that both this intermediate and the final complex bind transcription factor IIB in the second step of pol II pre-initiation complex assembly. A consequence of such a reactive intermediate is that the rate of assembly of transcriptionally competent pre-initiation complexes from bi-directionally bound TBP is greatly increased.

AB - Binding of the TATA-binding protein (TBP) to the 'TATA' sequences present in the promoters of eukaryotic class II genes is the first step in the sequential assembly of transcription pre-initiation complexes. Myriad structural changes, including severe bending of the DNA, accompany TBP-TATA complex formation. A detailed kinetic study has been conducted to elucidate the mechanistic details of TBP binding and DNA bending. The binding of Saccharomyces cerevisiae TBP to the adenovirus major late promoter (AdMLP) was followed in real-time through a range of temperatures and TBP concentrations using fluorescence resonance energy transfer (FRET) and stopped-flow mixing. The results of association and relaxation kinetics and equilibrium binding experiments were analyzed globally to obtain the complete kinetic and energetic profile of the reaction. This analysis reveals a complex mechanism with two intermediate species, with the DNA in the intermediates apparently bent similarly to the DNA in the final complex. TBP binding and DNA bending occur simultaneously through the multiple steps of the reaction. The first and third steps in this sequential process show nearly identical large increases in both enthalpy and entropy, whereas the middle step is highly exothermic and proceeds with a large decrease in entropy. The first intermediate is significantly populated at equilibrium and resembles the final complex both structurally and energetically. It is postulated that both this intermediate and the final complex bind transcription factor IIB in the second step of pol II pre-initiation complex assembly. A consequence of such a reactive intermediate is that the rate of assembly of transcriptionally competent pre-initiation complexes from bi-directionally bound TBP is greatly increased.

KW - DNA-protein interaction

KW - Fluorescence resonance energy transfer (FRET)

KW - TATAa

KW - TBP

KW - Transcription

UR - http://www.scopus.com/inward/record.url?scp=0033603388&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033603388&partnerID=8YFLogxK

U2 - 10.1006/jmbi.1999.2835

DO - 10.1006/jmbi.1999.2835

M3 - Article

VL - 289

SP - 1327

EP - 1341

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 5

ER -