| Summary: | The over-expression of the Saccharomyces cerevisiae pyruvate kinase gene (PYK1) is limited at the level of transcription and translation (Moore et al, 1990a). High levels of PYK1 mRNA are not tolerated by the transformants which grow up to 5.2-fold slower than the untransformed host strain. In addition, the transformants are genetically unstable, reverting at high frequency to fast growing cells via plasmid loss (Moore et al., 1990c). A novel experimental system was designed to facilitate the analysis of the translational regulation of the PYK1 gene. This system had two components designed to overcome the problems associated with the use of a multi-copy PYK1 plasmid. The first component was a PYK-lacZ reporter gene under the transcriptional control of the PGK1 promoter and terminator sequences. The translational regulation of the PYK1 gene could therefore be studied independently of transcription via -galactosidase assays. The PYK1 5'-leader regulated the translation of the reporter gene. A control reporter gene was also constructed containing a 'neutral' 5'-leader sequence. The reporter genes were used to confirm that the PYK1 5'-leader was both necessary and sufficient to mediate the translational regulation. The second component was a GAL-regulable multi-copy PAL-PYK plasmid. This gene is strongly induced by galactose but was found not to be tightly repressed by glucose. This lack of tight repression restricts the use of this plasmid in analyses. The over-expression of the PAL-PYK mRNA, however, did not impose the predicted dosage limitation on the PYK-lacZ reporter gene. It was shown that the sequence at the 5'-end of the PYK1 mRNA and/or the position of the 5'-cap relative to the translation start codon is critical to the function of the PYK1 5'-leader in mediating the translational regulation. The implications of this result on the mechanism of translational regulation are discussed.
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