The proper functioning of many biological processes and synthetic genetic networks depends on the precise tuning of expression levels of key protein components. With growing interests in eukaryotic hosts and the increasing complexity of networks in synthetic biology, there is a need for the expansion of the genetic toolbox, particularly for the bioprocessing and biosynthesis applications in the yeast Saccharomyces cerevisiae. The available control elements in yeast generally focus on the regulation of transcription through alternative promoter systems. Synthetic RNA-based control elements placed in the untranslated regions (UTRs) of transcripts have the ability to regulate the posttranscriptional mechanisms of translation initiation and transcript stability. Such posttranscriptional elements have the added advantage of being coupled to any promoter for enhanced control strategies. Two types of posttranscriptional elements were examined in this thesis. The first type is a class of RNA hairpins baring AGNN tetraloops that are cleaved by the S. cerevisiae RNase III enzyme Rnt1p. By locating these hairpins in the 3’ UTR of a transcript, the endonucleolytic cleavage due to Rnt1p activity resulted in the rapid degradation of the transcript. We developed two libraries of RNA hairpins based on the randomization of critical regions in Rnt1p substrates that affect the enzyme’s ability to associate and/or cleave the hairpin. The modulation of the strength of binding and cleavage by Rnt1p resulted in changes in the steady-state transcript levels and thus protein levels. Through integration of an aptamer into the stem of an Rnt1p hairpin, we were able to develop a riboswitch based upon the direction inhibition of Rnt1p cleavage through association of the ligand in the sites of cleavage. The second type of posttranscriptional elements examined is the placement of internal ribosome entry sites (IRESes) in the 5’ UTR that initiate translation independent of the 5’ cap through direct interaction with the ribosomal machinery. We propose that the activity of small sequential IRESes can be tuned through varying the complementarity with the 18S ribosomal RNA (rRNA) to advance the creation of yeast multicistronic vectors. The application of Rnt1p hairpins and IRESes provide a key tool in synthetic biology for the construction of complex genetic networks in yeast where the predictable tuning of gene expression is necessitated.
