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Sequence-function relationships in E. coli transcriptional regulation

Citation

Jones, Daniel Lawson III (2014) Sequence-function relationships in E. coli transcriptional regulation. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:06022014-165211576

Abstract

Understanding how transcriptional regulatory sequence maps to regulatory function remains a difficult problem in regulatory biology. Given a particular DNA sequence for a bacterial promoter region, we would like to be able to say which transcription factors bind there, how strongly they bind, and whether they interact with each other and/or RNA polymerase, with the ultimate objective of integrating knowledge of these parameters into a prediction of gene expression levels. The theoretical framework of statistical thermodynamics provides a useful framework for doing so, enabling us to predict how gene expression levels depend on transcription factor binding energies and concentrations. We used thermodynamic models, coupled with models of the sequence-dependent binding energies of transcription factors and RNAP, to construct a genotype to phenotype map for the level of repression exhibited by the lac promoter, and tested it experimentally using a set of promoter variants from E. coli strains isolated from different natural environments. For this work, we sought to ``reverse engineer'' naturally occurring promoter sequences to understand how variations in promoter sequence affects gene expression. The natural inverse of this approach is to ``forward engineer'' promoter sequences to obtain targeted levels of gene expression. We used a high precision model of RNAP-DNA sequence dependent binding energy, coupled with a thermodynamic model relating binding energy to gene expression, to predictively design and verify a suite of synthetic E. coli promoters whose expression varied over nearly three orders of magnitude.

However, although thermodynamic models enable predictions of mean levels of gene expression, it has become evident that cell-to-cell variability or ``noise'' in gene expression can also play a biologically important role. In order to address this aspect of gene regulation, we developed models based on the chemical master equation framework and used them to explore the noise properties of a number of common E. coli regulatory motifs; these properties included the dependence of the noise on parameters such as transcription factor binding strength and copy number. We then performed experiments in which these parameters were systematically varied and measured the level of variability using mRNA FISH. The results showed a clear dependence of the noise on these parameters, in accord with model predictions.

Finally, one shortcoming of the preceding modeling frameworks is that their applicability is largely limited to systems that are already well-characterized, such as the lac promoter. Motivated by this fact, we used a high throughput promoter mutagenesis assay called Sort-Seq to explore the completely uncharacterized transcriptional regulatory DNA of the E. coli mechanosensitive channel of large conductance (MscL). We identified several candidate transcription factor binding sites, and work is continuing to identify the associated proteins.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:gene regulation, transcriptional regulation, E. coli, thermodynamics models, noise, sort-seq
Degree Grantor:California Institute of Technology
Division:Engineering and Applied Science
Major Option:Applied Physics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Phillips, Robert B.
Thesis Committee:
  • Phillips, Robert B. (chair)
  • Elowitz, Michael B.
  • Roukes, Michael Lee
  • Winfree, Erik
Defense Date:30 May 2014
Record Number:CaltechTHESIS:06022014-165211576
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:06022014-165211576
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8467
Collection:CaltechTHESIS
Deposited By: Daniel Jones
Deposited On:05 Jan 2015 17:25
Last Modified:05 Jan 2015 17:25

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