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The effect of macromolecular crowding on single-round transcription by E. coli RNA polymerase

SangYoon Chung, Eitan Lerner, Yan Jin, Soohong Kim, Yazan Alhadid, Logan Wilson Grimaud, Irina X. Zhang, Charles M. Knobler, William M. Gelbart, Shimon Weiss
doi: https://doi.org/10.1101/218321
SangYoon Chung
1Department of Chemistry & Biochemistry, University of California Los Angeles,
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Eitan Lerner
1Department of Chemistry & Biochemistry, University of California Los Angeles,
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Yan Jin
1Department of Chemistry & Biochemistry, University of California Los Angeles,
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Soohong Kim
1Department of Chemistry & Biochemistry, University of California Los Angeles,
5The Broad Institute of MIT and Harvard, Cambridge, MA 02142
6Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
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Yazan Alhadid
1Department of Chemistry & Biochemistry, University of California Los Angeles,
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Logan Wilson Grimaud
1Department of Chemistry & Biochemistry, University of California Los Angeles,
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Irina X. Zhang
1Department of Chemistry & Biochemistry, University of California Los Angeles,
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Charles M. Knobler
1Department of Chemistry & Biochemistry, University of California Los Angeles,
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William M. Gelbart
1Department of Chemistry & Biochemistry, University of California Los Angeles,
2Molecular Biology Institute (MBI), University of California Los Angeles,
3California NanoSystems Institute, University of California Los Angeles,
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  • For correspondence: gelbart@chem.ucla.edu
Shimon Weiss
1Department of Chemistry & Biochemistry, University of California Los Angeles,
2Molecular Biology Institute (MBI), University of California Los Angeles,
3California NanoSystems Institute, University of California Los Angeles,
4Department of Physiology, University of California Los Angeles, CA 90095, USA
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  • For correspondence: sweiss@chem.ucla.edu
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ABSTRACT

Biological reactions in the cellular environment differ physicochemically from those performed in dilute buffer solutions due to, in part, slower diffusion of various components in the cellular milieu, increase in their chemical activities, and modulation of their binding affinities and conformational stabilities. In vivo transcription is therefore expected to be strongly influenced by the ‘crowdedness’ of the cell. Previous studies of transcription under macromolecular crowding conditions have focused mainly on multiple cycles of RNAP-Promoter associations, assuming that the association is the rate-determining step of the entire transcription process. However, recent reports demonstrated that late initiation and promoter escape could be the rate-determining steps for some promoter DNA sequences. The investigation of crowding effects on these steps under single-round conditions is therefore crucial for better understanding of transcription initiation in vivo. Here, we have implemented an in vitro transcription quenched-kinetics single-molecule assay to investigate the dependence of transcription reaction rates on the sizes and concentrations of crowders. Our results demonstrate an expected slowdown of transcription kinetics due to increased viscosity, and an unexpected enhancement in transcription kinetics by large crowding agents (at a given viscosity). More importantly, the enhancement’s dependence on crowder size significantly deviates from hard-sphere model (scaled-particle theory) predictions, commonly used for description of crowding effects. Our findings shed new light on how enzymatic reactions are affected by crowding conditions in the cellular milieu.

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Posted November 12, 2017.
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The effect of macromolecular crowding on single-round transcription by E. coli RNA polymerase
SangYoon Chung, Eitan Lerner, Yan Jin, Soohong Kim, Yazan Alhadid, Logan Wilson Grimaud, Irina X. Zhang, Charles M. Knobler, William M. Gelbart, Shimon Weiss
bioRxiv 218321; doi: https://doi.org/10.1101/218321
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The effect of macromolecular crowding on single-round transcription by E. coli RNA polymerase
SangYoon Chung, Eitan Lerner, Yan Jin, Soohong Kim, Yazan Alhadid, Logan Wilson Grimaud, Irina X. Zhang, Charles M. Knobler, William M. Gelbart, Shimon Weiss
bioRxiv 218321; doi: https://doi.org/10.1101/218321

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