The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGKI gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.Although the genetic code is degenerate for most amino acids, the choice of synonymous codons in both procaryotic and eucaryotic genes is far from random (9)(10)(11)17). The nonrandom codon usage patterns in the organisms examined show two prevailing characteristics: a strong positive correlation between the gene expression level and the degree of biased codon usage, and a similar relationship between codon usage of the highly expressed genes and the relative abundance of the corresponding tRNAs (17, 18; for recent reviews see references 4 and lla).In the yeast Saccharomyces cerevisiae, more than 96% of the amino acids in highly expressed genes are encoded by a set of 25 codons (2). The genes for all abundant proteins (e.g., glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase [PGK], and alcohol dehydrogenase) use these major codons almost exclusively throughout their sequences; codons recognized by minor tRNAs are avoided (reviewed in reference 4).In this report the function of biased codon usage in the highly expressed gene for PGK (PGKJ) was studied. PGKI coding sequence with a heterologous gene, the expression level decreases by one or two orders of magnitude. It has been shown that this dramatic difference in all cases correlates with a reduced steady-state mRNA level (3,27). These observations show that in addition to the regulatory elements flanking the structural gene, the coding sequence itself contains crucial information with regard to the expression level of this gene. The choice of codons could be one of the parameters affecting expr...