Mechanisms of non-Shine-Dalgarno translation initiation
The mechanism of translation initiation in bacteria was first examined in E. coli, where the presence of a Shine-Dalgarno site preceding the start codon leads to the initiation of translation in the proper reading frame. Now with thousands of sequenced bacterial genomes it was discovered that less than 1/2 of all bacterial protein coding genes are preceded by a Shine-Dalgarno site. Additionally, individual bacterial species including many cyanobacteria and bacteroidetes, lack Shine-Dalgarno sites in nearly 90% of their genes! We are therefore investigating the mechanisms of non-Shine-Dalgarno initiation by utilizing Caulobacter crescentus. Caulobacter contains Shine-Dalgarno sites in only 23.5% of its genes, has a doubling time of less than 2 hours, has well established genetic tools, and has a well annotated transcriptome. We are currently utilizing ribosome profiling, translation reporters, and in vitro reconstituted translation initiation assays to dissect the factors required for non-Shine-Dalgarno initiation in Caulobacter.
Sub-cellular organization of mRNA decay in bacteria
In eukaryotic cells, mRNA decay is often organized in ribonucleoprotein granules like RNA processing-bodies or stress granules. We found that bacteria can also make similar ribonucleoprotein granules that we termed bacterial ribonucleoprotein bodies (BR-bodies) composed of Ribonuclease E, protein components of the RNA degradosome, and RNA. These BR-bodies appear to be important for mRNA degradation and are assembled by liquid-liquid phase separation from the cytoplasm forming a compartment with high concentrations of the RNA degradosome and RNA. Here were's currently utilizing high-throughput mRNA decay assays and cell biology experiments to probe the role of these granules in mRNA decay.
RNA-mediated mechanisms of cell cycle-regulation
The Caulobacter cell cycle is controlled by a genetic and biochemical circuit that functions in space and time to control the the transcription of ~20% of the entire genome. We recently found that approximately half of these mRNAs contained translational control to regulate the timing of expression. We are interested in the regulatory logic of RNA-mediated control and how it is integrated with the cell cycle-regulatory circuit. Here were's currently utilizing high-throughput mRNA decay assays and cell biology experiments to probe the role of mRNA decay and translational control in the cell cycle-regulatory circuit.