Date of Award
Fall 2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular Biophysics and Biochemistry
First Advisor
Strobel, Scott
Abstract
Translation initiation across domains of life is a tightly regulated process, and functional RNA motifs are critical regulatory features. This dissertation largely focuses on developing a novel technique with which to quantify the effect these RNA elements have on translation initiation and on unraveling the functional requirements of these structures. I have leveraged RelE’s ribosome-dependent endonuclease activity to develop a quantitative assay for translationally regulated mRNAs and validated this method on two bacterial translational riboswitches. I have also demonstrated its applicability to eukaryotic systems by showing that RelE can measure isoform-driven differences in ribosome loading between yeast 5′ leader sequences. Moving beyond a gel-based readout, I have integrated RelE cleavage with next generation sequencing to examine the ligand responsiveness of more than 23,000 variants of the Pseudomonas aeruginosa Gdm-II riboswitch. Quantitative single and double mutant functional data revealed a finely-tuned expression platform and key positions that temper the switch’s sensitivity, dynamic range, and apparent cooperativity. Beyond the comprehensive mutational analysis of known motifs described here, RelE cleavage can also serve as validation of novel regulatory elements identified through high-throughput techniques. Application of this method to RNA thermometers, viral IRESes, variant riboswitches, and T-box RNAs among others will increase our understanding of the biology, evolution, and therapeutic potential of these elements.
Recommended Citation
Focht, Caroline, "Monitoring Translation Regulation by Structured RNA Elements" (2022). Yale Graduate School of Arts and Sciences Dissertations. 724.
https://elischolar.library.yale.edu/gsas_dissertations/724
