Plasmids were assembled in which the coding region of the pyruvate kinase (PYK) gene of Saccharomyces cerevisiae was replaced by that of the B-galactosidase (LacZ) gene from Escherichia coli. Analysis of the resultant, chimaeric transcripts from low copy number, centromeric plasmids indicated that this substitution caused a dramatic reduction in the steady-state level of the messenger RNA (mRNA). This fluctuation cannot be wholly accounted for by the 2-fold decrease in mRNA stability observed. This is consistent with the existence of a transcriptional Downstream Activation Site (DAS) within the PYK coding region, analogous to the DAS reported within the yeast phosphoglycerate kinase gene (PGK; Kingsman, S M et al. (1985) Biotech. Gen. Eng. Rev. 3, 377). At these low levels of heterologous gene expression, comparison of the distribution of PYK and PYK/LacZ transcripts across polysome gradients revealed no significant effect mediated by their striking disparity in codon usage. Nevertheless, upon increasing B-galactosidase mRNA levels, via manipulation of plasmid copy number, a distinct decline in ribosome loading was observed for the heterologous PYK/LacZ transcript which was not mirrored by either endogenous PYK transcripts or other yeast mRNAs of high (Ribosomal protein 1) or moderate (Actin) codon bias. However, high levels of the PYK/LacZ mRNA did affect the translation of an endogenous mRNA with poor codon bias (TRP2). The possible basis for this phenomenon is discussed.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Purvis IJ, Loughlin L, Bettany AJ, Brown AJ

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