Retention of transcription initiation factor σ70 in transcription elongation: Single-molecule analysis

Achillefs N. Kapanidis; Emmanuel Margeat; Ted A. Laurence; Sören Doose; Sam On Ho; Jayanta Mukhopadhyay; Ekaterine Kortkhonjia; Vladimir Mekler; Richard H. Ebright; Shimon Weiss

doi:10.1016/j.molcel.2005.10.012

Retention of transcription initiation factor σ⁷⁰ in transcription elongation: Single-molecule analysis

Achillefs N. Kapanidis, Emmanuel Margeat, Ted A. Laurence, Sören Doose, Sam On Ho, Jayanta Mukhopadhyay, Ekaterine Kortkhonjia, Vladimir Mekler, Richard H. Ebright, Shimon Weiss

Research output: Contribution to journal › Article › peer-review

120 Scopus citations

Abstract

We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and σ⁷⁰ contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, ∼70%-90%, of early elongation complexes retain σ⁷⁰ and that a determinant for σ⁷⁰ recognition in the initial transcribed region increases σ⁷⁰ retention in early elongation complexes. The results establish that a significant fraction, ∼50%-60%, of mature elongation complexes retain σ⁷⁰ and that a determinant for σ⁷⁰ recognition in the initial transcribed region does not appreciably affect σ⁷⁰ retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain σ⁷⁰, the half-life of σ⁷⁰ retention is long relative to the timescale of elongation, suggesting that some complexes may retain σ⁷⁰ throughout elongation.

Original language	English
Pages (from-to)	347-356
Number of pages	10
Journal	Molecular Cell
Volume	20
Issue number	3
DOIs	https://doi.org/10.1016/j.molcel.2005.10.012
State	Published - 11 Nov 2005
Externally published	Yes

Bibliographical note

Funding Information:
We thank Dr. Younggyu Kim for useful discussions, Dr. Nam Ki Lee for help during initial stages of the project, and Joanne Tang for editorial assistance. We also thank J. Armitage, B. Berks, P. Cook, J. McDonnell, A. Kolb, S. Wigneshweraraj, and K. Tokatlidis for helpful comments. This work was funded by Department of Energy grants 02ER63339 and 04ER63938 and National Institutes of Health grant GM069709-01A1 to S.W. and by National Institutes of Health grant GM41376 and a Howard Hughes Medical Institute Investigatorship to R.H.E.

Funding

We thank Dr. Younggyu Kim for useful discussions, Dr. Nam Ki Lee for help during initial stages of the project, and Joanne Tang for editorial assistance. We also thank J. Armitage, B. Berks, P. Cook, J. McDonnell, A. Kolb, S. Wigneshweraraj, and K. Tokatlidis for helpful comments. This work was funded by Department of Energy grants 02ER63339 and 04ER63938 and National Institutes of Health grant GM069709-01A1 to S.W. and by National Institutes of Health grant GM41376 and a Howard Hughes Medical Institute Investigatorship to R.H.E.

Funders	Funder number
National Institutes of Health	GM069709-01A1
U.S. Department of Energy	02ER63339, 04ER63938
National Institute of General Medical Sciences	R01GM041376

Access to Document

10.1016/j.molcel.2005.10.012

Cite this

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title = "Retention of transcription initiation factor σ70 in transcription elongation: Single-molecule analysis",

abstract = "We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and σ70 contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, ∼70%-90%, of early elongation complexes retain σ70 and that a determinant for σ70 recognition in the initial transcribed region increases σ70 retention in early elongation complexes. The results establish that a significant fraction, ∼50%-60%, of mature elongation complexes retain σ70 and that a determinant for σ70 recognition in the initial transcribed region does not appreciably affect σ70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain σ70, the half-life of σ70 retention is long relative to the timescale of elongation, suggesting that some complexes may retain σ70 throughout elongation.",

author = "Kapanidis, {Achillefs N.} and Emmanuel Margeat and Laurence, {Ted A.} and S{\"o}ren Doose and Ho, {Sam On} and Jayanta Mukhopadhyay and Ekaterine Kortkhonjia and Vladimir Mekler and Ebright, {Richard H.} and Shimon Weiss",

note = "Funding Information: We thank Dr. Younggyu Kim for useful discussions, Dr. Nam Ki Lee for help during initial stages of the project, and Joanne Tang for editorial assistance. We also thank J. Armitage, B. Berks, P. Cook, J. McDonnell, A. Kolb, S. Wigneshweraraj, and K. Tokatlidis for helpful comments. This work was funded by Department of Energy grants 02ER63339 and 04ER63938 and National Institutes of Health grant GM069709-01A1 to S.W. and by National Institutes of Health grant GM41376 and a Howard Hughes Medical Institute Investigatorship to R.H.E. ",

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T1 - Retention of transcription initiation factor σ70 in transcription elongation

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AU - Kapanidis, Achillefs N.

AU - Margeat, Emmanuel

AU - Laurence, Ted A.

AU - Doose, Sören

AU - Ho, Sam On

AU - Mukhopadhyay, Jayanta

AU - Kortkhonjia, Ekaterine

AU - Mekler, Vladimir

AU - Ebright, Richard H.

AU - Weiss, Shimon

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PY - 2005/11/11

Y1 - 2005/11/11

N2 - We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and σ70 contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, ∼70%-90%, of early elongation complexes retain σ70 and that a determinant for σ70 recognition in the initial transcribed region increases σ70 retention in early elongation complexes. The results establish that a significant fraction, ∼50%-60%, of mature elongation complexes retain σ70 and that a determinant for σ70 recognition in the initial transcribed region does not appreciably affect σ70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain σ70, the half-life of σ70 retention is long relative to the timescale of elongation, suggesting that some complexes may retain σ70 throughout elongation.

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