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DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation

Boedicker, James Q. and Garcia, Hernan G. and Johnson, Stephanie and Phillips, Rob (2013) DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation. Physical Biology, 10 (6). Art. No. 066005. ISSN 1478-3967. PMCID PMC3915735. doi:10.1088/1478-3975/10/6/066005. https://resolver.caltech.edu/CaltechAUTHORS:20131204-074505690

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Abstract

As the chief informational molecule of life, DNA is subject to extensive physical manipulations. The energy required to deform double-helical DNA depends on sequence, and this mechanical code of DNA influences gene regulation, such as through nucleosome positioning. Here we examine the sequence-dependent flexibility of DNA in bacterial transcription factor-mediated looping, a context for which the role of sequence remains poorly understood. Using a suite of synthetic constructs repressed by the Lac repressor and two well-known sequences that show large flexibility differences in vitro, we make precise statistical mechanical predictions as to how DNA sequence influences loop formation and test these predictions using in vivo transcription and in vitro single-molecule assays. Surprisingly, sequence-dependent flexibility does not affect in vivo gene regulation. By theoretically and experimentally quantifying the relative contributions of sequence and the DNA-bending protein HU to DNA mechanical properties, we reveal that bending by HU dominates DNA mechanics and masks intrinsic sequence-dependent flexibility. Such a quantitative understanding of how mechanical regulatory information is encoded in the genome will be a key step towards a predictive understanding of gene regulation at single-base pair resolution.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1088/1478-3975/10/6/066005DOIArticle
http://iopscience.iop.org/1478-3975/10/6/066005PublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3915735/PubMed CentralArticle
ORCID:
AuthorORCID
Johnson, Stephanie0000-0001-9487-8583
Phillips, Rob0000-0003-3082-2809
Additional Information:© 2013 IOP Publishing Ltd. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Received 21 August 2013. Accepted for publication 14 October 2013. Published 15 November 2013. We would like to dedicate this paper to the memory of Jon Widom, who was a fundamental creative force in the conception and implementation of this experiment. We are grateful to Bob Schleif and Tom Kuhlman for helpful discussions and technical assistance, to Reid Johnson for the HU-expressing strain used in this work, to Sankar Adhya for the HU purification protocol, and to both Reid Johnson and Sankar Adhya for advice and help on the HU expression and purification. HU was purified in collaboration with Jost Vielmetter and Angela Ho at the Caltech Protein Expression Center. This work was supported by the National Institutes of Health (awards no. DP1 OD000217 (HGG and RP), no. R01 GM085286, no. R01 GM085286-01S (HGG, JQB, and RP), and 1 U54 Ca143869 (Northwestern PSOC Center)), NSF graduate fellowship (SJ) and La Fondation Pierre Gilles de Gennes (RP). Author contributions: JQB, HGG, SJ and RP designed research; JQB, HGG, and SJ performed research; and JQB, HGG, SJ, and RP analyzed data and wrote the paper.
Funders:
Funding AgencyGrant Number
NIHDP1 OD000217
NIHR01 GM085286
NIHR01 GM085286-01S
NIH1 U54 CA143869
NSF Graduate FellowshipUNSPECIFIED
La Fondation Pierre Gilles de GennesUNSPECIFIED
Issue or Number:6
Classification Code:PACS: 87.14.G-, 87.80.-y, 87.15.Cc, 87.15.La, 87.14.E-, 87.15.B-
PubMed Central ID:PMC3915735
DOI:10.1088/1478-3975/10/6/066005
Record Number:CaltechAUTHORS:20131204-074505690
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20131204-074505690
Official Citation:DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation James Q Boedicker et al 2013 Phys. Biol. 10 066005
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:42814
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:04 Dec 2013 16:35
Last Modified:10 Nov 2021 16:28

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