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A quantitative analysis of genetic transcriptional and post-transcriptional regulation in Escherichia coli

  • Author(s): Kuhlman, Thomas Edward
  • et al.
Abstract

It is the goal of systems biology to understand the behavior of the whole in terms of knowledge of the parts. This is hard to achieve in many cases due to the difficulty of characterizing the many constituents and their complex web of interactions involved in biological systems. In this dissertation we report attempts to confront this difficulty by applying quantitative methods to experimentally characterize and theoretically model the web of interactions involved in transcriptional and post- transcriptional regulation in E. coli. In Chapters 1 and 2, Combinatorial Transcriptional Control of the Lactose Operon of Escherichia coli, we study the combinatorial control of the lac promoter by the regulators LacR and CRP. We are able to account for the combinatorial control of the lac promoter quantitatively. Specifically, our analysis indicates that the sensitivity of the inducer response results from LacR-mediated DNA looping, which is significantly enhanced by CRP. In Chapter 3, Autoregulation as a Strategy in Synthetic Reporter Constructs, we predict that autoregulation of a transcription factor used to control expression of a reporter gene can lead to significantly decreased sensitivity of the response of the reporter to inducer. We show that this is indeed the case by constructing and assaying the response of two autoregulated reporter systems using the transcription factors LacR and TetR. In Chapter 4, Quantitative Characteristics of Gene Regulation by Small RNA, we study quantitatively two classes of bacterial small RNAs (sRNA) in Escherichia coli. We demonstrate that sRNA provide a novel mode of gene regulation with characteristics distinct from those of protein-mediated gene regulation. These include a threshold-linear response with a tuneable threshold and a built-in capability for hierarchical cross talk. In Chapter 5, Noise and Silence in Small RNA Regulation, we study in detail noise properties of a gene targeted by an sRNA. We predict that sRNA-mediated gene silencing is efficient in attenuating noise. In contrast, the onset of gene expression is dominated by fluctuations, leading to noise induced phenotypic diversity. Using a plasmid-borne synthetic sRNA-target pair in E. coli, we present evidence for possible realization of the expected bi-modality in growing cells

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