Defining the Precision and Sequence Determinants of Protein Synthesis Rates

Abstract

Protein synthesis drives cellular proliferation and produces the primary effectors of biological function. Understanding how protein synthesis rates are controlled is therefore a central question of quantitative biology. In this work, I explore both the degree of precision in protein production and the mechanisms by which protein synthesis rates are encoded in genome sequences. First, I explore whether proportional synthesis of protein complex subunits, a phenomenon widely observed in bacterial protein synthesis, is a strategy adopted by eukaryotes. Using ribosome profiling, I observe proportional synthesis in the budding yeast Saccharomyces cerevisiae and through chromosomal duplications show that the precision in these protein synthesis rates is hard-coded without widespread negative feedback regulation. Proportional synthesis is also seen in large abundant complexes conserved in higher eukaryotes. I additionally present work to unify this understanding of proportional synthesis with evidence of post-translation buffering of protein complex subunit abundance derived from mass spectrometry. In the second half of this thesis, I turn my focus towards understanding how RNA decay and processing rates, two poorly understood processes central to the quantitative tuning of protein synthesis, are encoded in bacterial mRNAs. Using high-resolution RNA end-mapping techniques in combination with genetic perturbations to stabilize intermediates of RNA decay, I generate a global map of positions of endonuclease cleavage in Bacillus subtilis. Coupling this approach with knockouts of specific endonucleases, I greatly expand and refine the known set of targets of RNase Y, its specificity factor YlbF, and RNase III. Through this, I capture sequence and structural features recognized by RNase Y, the primary endonuclease initiating RNA decay in B. subtilis. I additionally provide evidence for a novel RNA 5′ end trimming activity of unknown origin. Finally, I present a system for massively parallel interrogation of the sequence-function relationship of B. subtilis mRNA processing sites. Using this approach, I uncover key sequence determinants of cggR-gapA and glnRA operon processing by RNase Y. Taken together, this work provides insight into the degree of precision achieved in gene expression across life and reveals mechanistic details of how protein synthesis can be tuned through RNA decay in a model Gram- positive bacterium.