Molecular dissection of the genetic mechanisms that underlie expression conservation in orthologous yeast ribosomal promoters

Abstract

Recent studies have shown a surprising phenomenon, whereby orthologous regulatory regions from different species drive similar expression levels despite being highly diverged in sequence. Here, we investigated this phenomenon by genomically integrating hundreds of ribosomal protein (RP) promoters from nine different yeast species into S. cerevisiae and accurately measuring their activity. We found that orthologous RP promoters have extreme expression conservation even across evolutionarily distinct yeast species. Notably, our measurements reveal two distinct mechanisms that underlie this conservation and which act in different regions of the promoter. In the core promoter region, we found compensatory changes, whereby effects of sequence variations in one part of the core promoter were reversed by variations in another part. In contrast, we observed robustness in Rap1 transcription factor binding sites, whereby significant sequence variations had little effect on promoter activity. Finally, cases in which orthologous promoter activities were not conserved could largely be explained by the sequence variation within the core promoter. Together, our results provide novel insights into the mechanisms by which expression is conserved throughout evolution across diverged promoter sequences.

Core promoter sequence in yeast is a major determinant of expression level

“…7), similar to what we recently observed at the level of the entire RP promoter region (Zeevi et al 2014). These results suggest that in order for orthologous sensu stricto RP promoters to maintain their promoter activity during evolution, they had to maintain the activity of their core promoter, and the alternative option whereby their core promoter activity might have diverged and be compensated for by other mechanisms is ruled out.…”

“…In each plasmid, the 118-bp-long core promoter region was upstream of an intact YFP sequence and downstream from the [−528,−129] region (positions relative to the translation start site) of the RPL28 promoter, a constitutively expressed ribosomal protein (RP) promoter that includes a tandem pair of binding sites for the Rap1 transcription factor, the main regulator of yeast RP promoters (Lieb et al 2001), as well as binding sites for Fhl1 and Sfp1, which are also known to regulate RP gene expression level (Zeevi et al 2014). This region of the RPL28 promoter does not contain TATA elements downstream from the Rap1 sites, limiting its competition with the core promoter variant over PIC recruitment.…”

“…Core promoter activity is highly correlated to the activity of the entire promoter. (A) A comparison of our core promoter activity measurements (x-axis) to previously measured promoter activities (y-axis) for 238 Saccharomyces sensu stricto RP promoters (Zeevi et al 2014) reveals a high correlation between the two measures. Dark red dots mark promoters that are not regulated by Rap1.…”

“…The RP core promoters of the Saccharomyces sensu stricto species tend to be more conserved than upstream promoter regions, yet they too substantially diverged, with some parts showing even <70% sequence identity between S. cerevisiae and S. bayanus (Zeevi et al 2014). In our library, we included the [−118,−1] regions of orthologous RP promoters representing four sensu stricto species and 67 genes.…”

“…One such set of 238 core promoters originates from orthologous ribosomal protein promoters of four Saccharomyces sensu stricto species (S. cerevisiae, S. paradoxus, S. mikatae and S. bayanus), representing 67 distinct RP genes, for which we recently measured the activity of the entire promoter within S. cerevisiae (see Supplemental Table 3; Zeevi et al 2014). Notably, we found a very high correlation between our measurements of the activity of the core promoter and the activity reported for the entire promoter (r = 0.78, P < 10 −49 ) ( Fig.…”