RNA Roadblocks: Hairpins for Protein Level Tuning in S. Cerevisiae.

To modulate protein levels in eukaryotes, gene expression is normally modified at the transcriptional level by exchanging or mutating the promoter of a gene of interest. Unfortunately this approach often also alters the regulation of a protein as promoter sequences encode information both on the strength of expression and when expression occurs. To enable control over protein levels without modifying promoters in S. cerevisiae, we introduce here a novel method for tuning the efficiency of protein translation. By placing strong hairpins in the 5’ UTR of expressed mRNAs we show here that we can down-tune translation. We use the predicted free energy of folding of the 5’UTR to estimate the final expression level of the protein in question.

We designed and constructed a library of secondary structures, calculating the free energy for every possible member, optimizing the design to obtain a wide spread of folding energies with only minor changes to 5’UTR sequences. We characterized several libraries by expression of green fluorescent protein and went on to investigate the further effect of 5’UTR length and the location of the hairpin within the 5’UTR. We then applied this approach to modulate expression from 4 different constitutive promoters and from the GAL1 promoter, without having to disrupt the tight regulation of the native promoter. This work thus provides a new class of modular part for manipulating eukaryotic gene expression, and by following RNA design principles stemming from free energy calculations, allows predictable tuning of protein production.