A Genome-Wide Activity Assessment of Terminator Regions in Saccharomyces cerevisiae Provides a ″Terminatome″ Toolbox

Abstract

The terminator regions of eukaryotes encode functional elements in the 3' untranslated region (3'-UTR) that influence the 3'-end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. However, the contribution of these terminator regions to gene expression remains unclear, and therefore their utilization in metabolic engineering or synthetic genetic circuits has been limited. Here, we comprehensively evaluated the activity of 5302 terminator regions from a total of 5880 genes in the budding yeast Saccharomyces cerevisiae by inserting each terminator region downstream of the P TDH3 - green fluorescent protein (GFP) reporter gene and measuring the fluorescent intensity of GFP. Terminator region activities relative to that of the PGK1 standard terminator ranged from 0.036 to 2.52, with a mean of 0.87. We thus could isolate the most and least active terminator regions. The activities of the terminator regions showed a positive correlation with mRNA abundance, indicating that the terminator region is a determinant of mRNA abundance. The least active terminator regions tended to encode longer 3'-UTRs, suggesting the existence of active degradation mechanisms for those mRNAs. The terminator regions of ribosomal protein genes tended to be the most active, suggesting the existence of a common regulator of those genes. The ″terminatome″ (the genome-wide set of terminator regions) thus not only provides valuable information to understand the modulatory roles of terminator regions on gene expression but also serves as a useful toolbox for the development of metabolically and genetically engineered yeast.

A genome-wide resource for high-throughput genomic tagging of yeast ORFs

“…2d). Consistent with these observations, the heterologous ADH1 terminator used in mNG-II and mSC-II libraries is stronger than terminator sequences of most yeast ORFs 18 . Together these results demonstrate that tagging modules with heterologous terminators, commonly used for C-terminal protein tagging in yeast 19,20 , can measurably impact protein expression and suggest applications of the C-SWAT library to study regulation of gene expression.…”

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

“…Although the constructs were controlled by the same promoter/terminator systems for both the yeast and E. faecalis modules (Fig. 2a), differences in mRNA levels may be due to stability of different transcripts (especially considering that the E. faecalis genes are not native genes while the yeast genes are native), or to idiosyncratic characteristics of the ORFs/constructs and their compatibility with specific promoters and terminators (Carquet et al, 2015;Yamanishi et al, 2013). Regardless of the mechanism, these data demonstrate that expression of the yeast genes is limited at the RNA level relative to the E. faecalis genes.…”

Short Synthetic Terminators for Improved Heterologous Gene Expression in Yeast

“…Nevertheless, recent work in S. cerevisiae has aptly demonstrated that varying the choice of native terminators can influence mRNA half-life of heterologously expressed genes and result in increased or decreased protein levels (5)(6)(7) . The underlying force responsible for these variations in gene expression is disparate stabilizing elements found in different native terminators (2,8) .…”