A systems approach to understand translation and sRNA-mediated gene regulation in

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DESCRIPTION (provided by applicant): Cell differentiation in Caulobacter crescentus occurs as a function of the cell cycle, where transcriptional circuitry guides progression through distinct phases of gene expression to ensure multiple events take place in a spatiotemporally regulated manner. Despite compelling evidence of the regulation of translation by the cell cycle circuitry, the role of translation is relatively unexplored in Caulobacter cell cycle progression. The goal of this project is to understand how gene expression is regulated by mRNA translation to ensure cell cycle progression in Caulobacter. In order to understand this process, I will employ a new method, ribosome profiling, to monitor genome wide changes in translation by measuring the number of ribosome-protected mRNA fragments as well as ribosome positions with high throughput sequencing. Additionally, I will explore the role of a new class of genes, trans encoded small RNAs, which were recently discovered to be expressed during distinct phases of the cell cycle. This class of genes functions by base pairing to mRNAs through the RNA chaperone Hfq and positively or negatively regulating mRNA translation and mRNA stability. I will use ribosome profiling with knock downs of genes encoding small RNAs and Hfq to identify how these genes aid in translation regulation. Additionally, I will determine which sRNAs and their mRNA targets physically associate with the regulator Hfq, and determine the Hfq binding sites on the RNAs by using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). This method UV crosslinks Hfq ribonucleoprotein complexes in vivo, then uses a ribonuclease protection to capture the bound fragments, and uses high-throughput sequencing to identify the sRNA and mRNA binding sites of Hfq. With this broad approach, I will determine how global mRNA translation is regulated to ensure the dynamic changes in gene expression that drive cell cycle progression in Caulobacter.