UC Berkeley

Bacterial survival depends on the ability of cells to rapidly adapt to diverse environments, stress and pathogenesis. Many of these adaptive pathways involve regulation by small non-coding RNAs (sRNAs) that act in trans by binding to the 5' untranslated region of target mRNAs to form sRNA-target mRNA duplexes that alter mRNA translation and/or degradation. This dissertation examines the regulation of gene expression by sRNAs using synthetic gene circuits in Escherichia coli to understand their regulatory properties and to gain insight into why sRNAs may have evolved as critical regulators of bacterial stress responses.

In the first chapter, we characterize the regulatory properties of sRNAs in vivo and compare them to that of transcription factors. Specifically, we examine the dose response curves and dynamics of regulating gene expression with sRNAs and transcription factors and find that the primary advantage of sRNAs are that their activity can be rapidly turned off. This can provide an advantage for cells needing to signal and reprogram gene expression rapidly in response to environmental stress. In the second chapter, we examine sRNA regulation in the context of a network controlled by competition for the Hfq protein, which mediates the activity of most sRNAs. The role of Hfq is critical in bacterial stress responses because it allows efficient gene regulation by sRNAs in response to cellular stress including during starvation, and shifts in optimal pH, osmolarity, and temperature. We show that physiological levels of Hfq are low and that this can lead to reduced activity of sRNAs and cross-talk between different sRNAs and target mRNAs due to competition for Hfq. We further show that this competition can be at least partly relieved when the sRNAs and target mRNAs are co-transcribed, which would allow more efficient duplex formation. In the third chapter, we characterized sRNA regulation using the fluorescent in situ hybridization (RNA FISH) technique. We showed that RNA FISH can allow rapid and broad characterization of a large number of sRNA-target mRNA pairs and that thereby provide information (such as the localization, cell-to-cell variation, and intracellular variation of sRNAs and target mRNAs) that cannot be obtained by other techniques. Together, the three chapters provide insight into the roles of sRNAs and their networks during bacterial stress responses, and suggest paths for future exploration.