Regulating expression of mistranslating tRNAs by readthrough RNA polymerase II transcription

BioRxiv : the Preprint Server for Biology
Matthew D BergChristopher J Brandl

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 mis-incorporates 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 G1 phase. These cells are impermeable to propidium iodide, yet ...Continue Reading

Datasets Mentioned

BETA
PXD028496

Methods Mentioned

BETA
Flow Cytometry
FACS
reverse-phase chromatography
two hybrid

Software Mentioned

FlowJo Flow Cytometry Analysis
ZEN Blue Pro
R package “ growthcurver ”
ImageJ
Percolator
Comet

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