Bacterial Regulatory Mechanisms of Gene Expression During Stress Responses with Focus on Closely Spaced Promoters

Vatsala Chauhan

    Research output: Book/ReportDoctoral thesisCollection of Articles

    Abstract

    Bacteria are exposed to changing environments and other stresses, such as antibiotics. Some of these events can even be lethal. Their phenotypic adaptations to these stresses are driven by internal mechanisms of gene regulation that, therefore play a fundamental role in their survivability. The core mechanism of gene regulation is arguably the promoter regions. These are DNA sequences that largely determine whether a gene is sensitive to specific transcription factors, supercoiling fluctuations, and other regulatory factors and events.

    In this thesis, we used Escherichia coli as a model organism to study bacterial mechanisms of genome-wide expression regulation during stresses, focusing on the promoters in tandem formation. For that, we started by developing a novel method to determine single-cell distributions of RNA numbers from flow cytometry data. This method can predict the moments of the distribution of the single-cell RNA numbers from the moments of the distribution of total fluorescence of cells expressing the proteins that the RNAs code for. This greatly facilitates the study of transcription dynamics using large numbers of cells as a source of data.

    Next, we used a large strain library of tagged genes controlled by tandem promoters. From the single-cell distributions of their protein levels under different stress conditions, we dissected the main features controlling the kinetics of overlapping tandem promoters. Specifically, we identified the distance between start sites and the dynamics of the transcription initiation at each promoter, as the main factors.

    Finally, we designed and constructed a strain library of synthetic genes controlled by non-overlapping tandem promoters. We used them to validate, by proof of concept, that they can be used to engineer genes with predictable dynamics. Moreover, we identified a key variable controlling these constructs, namely, the strength of the downstream promoter, which acts as the main limiting factor of the overall transcription rate. Overall, this study dissected important regulatory features of tandem promoters. The findings facilitate their use as building blocks of future synthetic circuits.
    Original languageEnglish
    Place of PublicationTampere
    ISBN (Electronic)978-952-03-3112-2
    Publication statusPublished - 2023
    Publication typeG5 Doctoral dissertation (articles)

    Publication series

    NameTampere University Dissertations - Tampereen yliopiston väitöskirjat
    Volume886
    ISSN (Print)2489-9860
    ISSN (Electronic)2490-0028

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