Transcriptional control is mediated by batteries of transcription factors that interact with DNA, each other and with the transcription apparatus itself. In many of these cases, proteins bound to regulatory regions hundreds to thousands of basepairs away from the basal transcription apparatus perform a kind of biological action at a distance by binding at more than one site on the DNA simultaneously resulting in wholesale rearrangements of the genomic DNA (e.g. DNA looping). However, the mechanistic underpinnings of transcriptional control, especially that involving large-scale deformations of the DNA, and the quantitative consequences of this control largely remain to be elucidated. The work proposed here builds upon three distinct but related pillars to analyze the formation of these complexes: i) the use of statistical mechanics models to compute the relation between regulatory architecture and the level of gene expression, ii) the use of single-molecule tethered particle experiments to measure the stability and kinetics of formation of transcription factor-DNA complexes and iii) the measurement of level of gene expression in living cells. In all three of these cases, DNA sequence (flexibility), binding site strength and number of operators are used as dials to tune the level of gene expression.

Public Health Relevance

Transcriptional regulation is at the heart of processes in biology ranging from the formation of biofilms to cell differentiation to evolution. In its role as one of the central threads in the study of genes and how they are regulated, transcriptional control is relevant to biology and medicine alike. The work proposed here aims to strengthen our understanding of transcription in microbes in a way that will have applications from synthetic biology to the study of pathogens.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM085286-04
Application #
8215827
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Lewis, Catherine D
Project Start
2009-02-17
Project End
2013-08-31
Budget Start
2012-02-01
Budget End
2013-08-31
Support Year
4
Fiscal Year
2012
Total Cost
$337,400
Indirect Cost
$129,129
Name
California Institute of Technology
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
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Jones, Daniel L; Brewster, Robert C; Phillips, Rob (2014) Promoter architecture dictates cell-to-cell variability in gene expression. Science 346:1533-6
Brewster, Robert C; Weinert, Franz M; Garcia, Hernan G et al. (2014) The transcription factor titration effect dictates level of gene expression. Cell 156:1312-23
Rydenfelt, Mattias; Cox 3rd, Robert Sidney; Garcia, Hernan et al. (2014) Statistical mechanical model of coupled transcription from multiple promoters due to transcription factor titration. Phys Rev E Stat Nonlin Soft Matter Phys 89:012702
Ngo, Huu B; Lovely, Geoffrey A; Phillips, Rob et al. (2014) Distinct structural features of TFAM drive mitochondrial DNA packaging versus transcriptional activation. Nat Commun 5:3077
Johnson, Stephanie; van de Meent, Jan-Willem; Phillips, Rob et al. (2014) Multiple LacI-mediated loops revealed by Bayesian statistics and tethered particle motion. Nucleic Acids Res 42:10265-77
Boedicker, James Q; Garcia, Hernan G; Johnson, Stephanie et al. (2013) DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation. Phys Biol 10:066005
Boedicker, James Q; Garcia, Hernan G; Phillips, Rob (2013) Theoretical and experimental dissection of DNA loop-mediated repression. Phys Rev Lett 110:018101
Garcia, Hernan G; Lee, Heun Jin; Boedicker, James Q et al. (2011) Comparison and calibration of different reporters for quantitative analysis of gene expression. Biophys J 101:535-44
Sanchez, Alvaro; Garcia, Hernan G; Jones, Daniel et al. (2011) Effect of promoter architecture on the cell-to-cell variability in gene expression. PLoS Comput Biol 7:e1001100

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