Figure 1.

The promoter reference technique. A, PRT design: two identical transcriptional promoters expressing, respectively, a test protein and a reference protein. B, identical sets of DNA elements, developed by Kötter et al. (80), that are present at the two identical, tandem P_TDH3 promoters in the current version of PRT. C, a “generic” PRT plasmid, derived from the previously described pJO629 plasmid (54, 55). It contains DNA elements that enable propagation of this plasmid in E. coli (including the ampicillin-encoding amp gene and the origin of DNA replication from the E. coli plasmid pBR322) and in S. cerevisiae (as a low-copy plasmid), including the HIS3 marker gene, the replication/segregation CEN-ARS DNA segment, and the CYC1-derived transcription-terminating DNA segment. This plasmid also encodes the flag/ha-tagged _fDHFR_ha reference protein and a C-terminally triple-flag-tagged test protein. In the present study, the test proteins were X-Mdh2_3f bearing either N-terminal Pro or N-terminal Ser, as described under “Results and discussion” (Fig. 3 and Fig. S1). The test protein and the reference protein are expressed from two identical P_TDH3 promoters containing additional DNA elements (80). Once transcribed into an mRNA, these elements form 5′-RNA aptamers that can bind to Tc, thereby repressing translation of that mRNA in cis.