Uncovering the Mechanism of Intrinsic Transcription Termination in Order to Develop Point-of- Care Diagnostics.

Verosloff, Matthew  Stuart

doi:https://doi.org/10.21985/n2-w9sj-ta86

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Uncovering the Mechanism of Intrinsic Transcription Termination in Order to Develop Point-of- Care Diagnostics.

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Regulatory RNAs are found throughout nature controlling critical cellular processes and enabling cells to sense and respond to their environment. In order to provide genetic regulation, these RNAs can selectively bind to target molecules, proteins, and invading pathogens, all while modulating gene expression on both the transcriptional and translational level. They are capable of carrying out these critical tasks for maintaining homeostasis. RNAs carry out these complex tasks despite their relatively simplistic composition, canonically comprised of only four nucleotide building blocks. In spite of previous work modeling these systems, the mechanism by which regulatory RNA’s sequence and structural elements are able to enable their diverse functionality remains unclear. To address this gap, we set out to develop novel ways to interrogate the relationship between the sequence and structural elements of an RNA, and its function. We started by probing RNA elements that specifically regulate intrinsic termination in prokaryotes. To accomplish this, we developed reporter libraries by strategically varying previously unexplored regions of thenascent RNA, in order to analyze their contribution to the overall mechanism of termination. Resulting impacts to intrinsic termination efficiency were measured in vivo and classified by principal component analysis and regression tree analysis. In this manner, we demonstrated that limited secondary structure and intramolecular base stacking within the RNA regions immediately upstream of the terminator hairpin correlates with efficient termination. This led to our working hypothesis that the elements in this upstream region are required to enhance the speed of the formation of the terminator hairpin to enable it to function on the kinetic scale required by prokaryotic intrinsic termination. We next moved to studying the termination mechanism of the eukaryotic RNA Polymerase III. This mechanism had previously been the source of debate as there is a lack of consensus on the precise nascent RNA elements that are required for efficient termination at intended loci. Various research groups have shown differing results from in vitro experimentation on whether the presence of upstream structure within the nascent RNA has an impact on the efficiency of Pol III termination. We addressed this by developing a novel in cellulo assay within the human cancer cell line HEK293FT. This assay allowed us to quantify transcriptional termination efficiency caused directly by RNA elements upstream of a transcriptionally insulated fluorescent reporter. In this manner, we provided support for the hypothesis that Pol III termination requires both an RNA sequence comprised of a poly-uracil tract along with upstream secondary structure. While termination could occur at poly-uracil tracts longer than those found on average throughout the human genome, robust termination at shorter but relevant tracts required a structural component. We also found that this effect weakens as the structural element is moved further away from the poly-uracil tract. Following elucidation of these termination mechanisms, we were interested in whether these naturally derived principles could be used to forward-design synthetic RNAs encoded to carry out a desired task. Because cells utilize regulatory RNA to detect the presence of both pathogens and environmental contaminants, we hypothesized that we could also use our synthetically derived variants for a similar purpose. To this point, we demonstrated that synthetic intrinsic terminators, conditionally active in the presence of a trans-acting small RNA (sRNA), could indeed be developed with the requisite degree of termination efficiency. Furthermore, these novel terminators possessed the necessary characteristics for empowering viral diagnostics. We integrated these synthetic RNA sensors into a schematic for our point-of-care diagnostic PLANTdx, which provides the molecular sensing and computation needed to report on the presence or absence of plant pathogens in a sample lysate. This work highlights how further insights into the natural regulation of RNA will also provide the means for improving upon current biology-based applications. Overall, these studies demonstrate steps towards a complete understanding of intrinsic termination spanning across the domains of life. It is our hope that the methodology and suggestions for future directions highlighted within this work will lead to a complete and accurate model for all variants of intrinsic termination. We were especially excited that the insights gleamed within our analysis were further verified by our capacity to utilize them as design rules for the development of synthetic terminators. These design rules enabled the creation of novel userfriendly diagnostics, demonstrating needed improvements to current golden standard technology. This work showcases the capacity of basic biology research to not only further our understanding of the natural world but also to provide the basis for the production of next generation biology based applications. Therefore, this research along with future experimentation will work towards improving both our planet and living standards for all humanity.

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