Characterizing fundamental principles of translational control in the bacterial model organism E. coli using a novel deep-sequencing technique

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Gene expression, the most fundamental process in living cells, is carried out in two steps: transcription of the DNA sequence of a gene into mRNA by RNA polymerase (RNAP), and translation of the mRNA template into protein by ribosomes. In bacteria, both steps are coupled as ribosomes initiate translation of mRNA molecules during their transcription (transcription-translation coupling).Advances in sequencing technology (deep sequencing) transformed our understanding of translational and transcriptional control by providing a genome-wide view of the positions of individual translating ribosomes on mRNAs, and transcribing RNAPs. This enabled us to determine what proteins are synthesized and how much of each, as well as where the ribosome and RNAP are pausing. However, these methods cannot measure distances between machineries on single mRNA molecules. Thus, we do not know the spacing between multiple ribosomes translating an mRNA molecule, or between the lead ribosome and transcribing RNAP. Therefore, we lack fundamental parameters to describe and understand translation and transcription-translation coupling.I am developing a novel deep-sequencing method (ASITE) that allows us to measure both the exact positions of ribosomes and their spacing relative to each other and to RNAP on individual mRNAs in E. coli. Using ASITE, we will ask: Is translation initiation stochastic, or does it occur in bursts with packs of ribosomes initiating together? Do mRNAs regulate ribosome spacing during translation, and how? Do cells coordinate ribosome and RNAP distances, and if yes, what are the mechanisms to do so? These studies will likely have a transformative impact on understanding translation in bacteria at the single mRNA level.Furthermore, the ASITE method developed here will be widely applicable to translation research in eukaryotic organisms, including human cells and viral translation programs.