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
Garcia, Hernan G; Brewster, Robert C; Phillips, Rob (2016) Using synthetic biology to make cells tomorrow's test tubes. Integr Biol (Camb) 8:431-50
Lovely, Geoffrey A; Brewster, Robert C; Schatz, David G et al. (2015) Single-molecule analysis of RAG-mediated V(D)J DNA cleavage. Proc Natl Acad Sci U S A 112:E1715-23
Kreamer, Naomi N; Phillips, Rob; Newman, Dianne K et al. (2015) Predicting the impact of promoter variability on regulatory outputs. Sci Rep 5:18238
Phillips, Rob (2015) Napoleon Is in Equilibrium. Annu Rev Condens Matter Phys 6:85-111
Mulligan, Peter J; Chen, Yi-Ju; Phillips, Rob et al. (2015) Interplay of Protein Binding Interactions, DNA Mechanics, and Entropy in DNA Looping Kinetics. Biophys J 109:618-29
Phillips, Rob (2015) Theory in Biology: Figure 1 or Figure 7? Trends Cell Biol 25:723-9
Razo-Mejia, M; Boedicker, J Q; Jones, D et al. (2014) Comparison of the theoretical and real-world evolutionary potential of a genetic circuit. Phys Biol 11:026005
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
Weinert, Franz M; Brewster, Robert C; Rydenfelt, Mattias et al. (2014) Scaling of gene expression with transcription-factor fugacity. Phys Rev Lett 113:258101

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