Regulating Expression of Mistranslating tRNAs by Readthrough RNA Polymerase II Transcription

Abstract

Transfer RNA (tRNA) variants that alter the genetic code increase protein diversity and have many applications in synthetic biology. Since the tRNA variants can cause a loss of proteostasis, regulating their expression is necessary to achieve high levels of novel protein. Mechanisms to positively regulate transcription with exogenous activator proteins like those often used to regulate RNA polymerase II (RNAP II)-transcribed genes are not applicable to tRNAs as their expression by RNA polymerase III requires elements internal to the tRNA. Here, we show that tRNA expression is repressed by overlapping transcription from an adjacent RNAP II promoter. Regulating the expression of the RNAP II promoter allows inverse regulation of the tRNA. Placing either Gal4- or TetR–VP16-activated promoters downstream of a mistranslating tRNASer variant that misincorporates serine at proline codons in Saccharomyces cerevisiae allows mistranslation at a level not otherwise possible because of the toxicity of the unregulated tRNA. Using this inducible tRNA system, we explore the proteotoxic effects of mistranslation on yeast cells. High levels of mistranslation cause cells to arrest in the G1 phase. These cells are impermeable to propidium iodide, yet growth is not restored upon repressing tRNA expression. High levels of mistranslation increase cell size and alter cell morphology. This regulatable tRNA expression system can be applied to study how native tRNAs and tRNA variants affect the proteome and other biological processes. Variations of this inducible tRNA system should be applicable to other eukaryotic cell types.

Genetic background and mistranslation frequency determine the impact of mistranslating tRNASerUGG

“…Yeast cells expressing tRNA Ser UGG,G26A , which contains a proline anticodon, mistranslate serine at proline codons ( Berg, Zhu, et al 2019 ; Berg, Isaacson, et al 2021 ). The G26A mutation is required in combination with the anticodon change to dampen otherwise lethal levels of mistranslation ( Berg et al 2017 ).…”

“…To regulate the frequency of mistranslation, we took advantage of our finding that placing a GAL1 promoter ( GAL1pr ) sequence up or downstream of a tRNA gene represses tRNA expression when cells are grown in galactose ( Berg, Isaacson, et al . 2021 ).…”

“…Previously, using mass spectrometry, we determined the frequency of proline to serine mistranslation in strains containing centromeric plasmids expressing wild-type tRNA Ser , tRNA Ser UGG,G26A , tRNA Ser UGG,G26A with 5′ GAL1pr , and tRNA Ser UGG,G26A with 3′ GAL1pr in galactose containing medium to be 0.3%, 5.6%, 3.5%, and 0.9%, respectively ( Fig. 3a ; Berg, Isaacson, et al . 2021 ).…”

“…URA3- containing centromeric plasmids expressing tRNA Ser (pCB3076), tRNA Ser UGG,G26A (pCB4023), tRNA Ser UGG,G26A with 5′ GAL1pr (pCB4568), and tRNA Ser UGG,G26A with 3′ GAL1pr (pCB4566) are described in Berg et al (2017) and Berg, Isaacson, et al (2021 ).…”

“…When mistranslation reaches a threshold, protein quality control mechanisms no longer protect the cell and growth is impaired ( Berg, Zhu, et al 2019 ). The extent of growth impairment is inversely related to the frequency of mistranslation in a linear fashion with yeast growth being arrested when mistranslation approaches approximately 12% ( Berg, Isaacson, et al . 2021 ).…”