Implications and Applications of Transfer RNA Variants that Mistranslate the Genetic Code
Abstract
Genetic information is passed from DNA to RNA to protein through the processes of transcription and translation. Transfer RNAs (tRNA) are the adaptors that bring amino acids to the growing polypeptide chain during translation and decode the three base codons that define protein sequence. Mistranslation occurs when an amino acid different from what is specified by the genetic code is inserted into a protein. tRNA variants cause mistranslation by decreasing the accuracy of amino acid charging or by altering decoding at the ribosome. My goal was to characterize mistranslating tRNA variants, identify their effects on cells and determine mechanisms used to cope with the resulting proteotoxicity.
First, I focus on anticodon variants of Saccharomyces cerevisiae tRNASer. In isolation these are lethal due to a toxic level of mistranslation, but using a genetic suppression assay, I identify second site mutations in tRNASer that decrease function and allow viable mistranslation levels. I propose that for mistranslating tRNAs to arise, cells first acquire an “ambivalent intermediate” mutation that decreases tRNA function. I characterize additional mutations in tRNASer that allow different mistranslation frequencies at a variety of non-serine codons. To further regulate the toxicity of mistranslation tRNAs and allow high levels of mistranslation, I develop an inducible tRNA expression system, negatively regulated by an RNA polymerase II promoter downstream of the tRNA. My studies demonstrate a mechanism by which mistranslating tRNA variants arise, factors that determine their toxicity and how mistranslation can be engineered for applications in synthetic biology.
Next, I investigate naturally occurring tRNA variants in human populations. Using a custom DNA capture array for the 610 human tRNA genes and a novel bioinformatics pipeline to accurately map variants, I identify ~66 tRNA variants per person including potential mistranslating variants and loss of function variants that could alter tRNA pools. I then perform a genetic screen in yeast demonstrating that mistranslation synergistically exacerbates growth defects caused by loss of a variety of genes, including those involved in protein quality control and actin cytoskeleton. I hypothesize that tRNA variation is a modulator of disease, particularly diseases characterized by loss of proteostasis.