Defining the Constellation of RNA Elements That Associate with Bacillus Subtilis HFQ
Dambach, Michael David
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Bacteria utilize a wide variety of genetic regulatory strategies in order to sense and respond to various environmental fluctuations in nutrient availability, temperature, salinity, and oxygen among others. As such, bacterial species have evolved highly coordinated and tightly regulated systems as a means of efficiently responding to potentially deleterious changes in environmental conditions. Traditionally DNA binding transcriptions factors were thought to be the primary means by which the cell executes a selective genetic response. However, the advent of microarray and next generation sequencing platforms, coupled with the wealth of sequenced genomes and powerful bioinformatics have revealed that RNA mediated post transcriptional gene regulation is wide spread in bacterial species and may in fact rival protein based regulatory systems in scope and breadth. RNA mediated post transcriptional gene regulation is broadly divided into two categories-those in which the RNA element is transcribed with the mRNA it regulates (cis-acting regulatory RNAs) or those which are transcribed independently from the gene that they regulate (trans-acting regulatory RNAs). In general cis-acting RNA elements are embedded within a 5' UTR of a gene that they regulate and may or may not require a protein cofactor to execute genetic regulation. Whereas, trans-acting regulatory RNAs, also known as sRNAs, function via base pairing with their target mRNA and this usually requires the protein chaperone Hfq. Hfq mediated gene regulation is poorly understood in Gram-positive organism, thus I undertook studies of this protein in the model Gram-positive organism Bacillus subtilis. I used co-immunoprecipitation and deep-sequencing to define the suite of RNA elements that associate with this regulatory protein. In addition I performed global transcriptomic studies on an Hfq deletion mutant in order to identify genes that are regulated via Hfq. These studies identified sRNAs that may be involved with sporulation. This led me to analyze the transcriptomic profile of Bacillus subtilis spores in an attempt to identify new sRNA regulators.