Yeast ENO1 promoter was prepared by a chemical synthetic method. Two variant promoters containing a pyrimidine-rich region (CT block), located between the TATA box and the transcription start site, either 32 or 34 base pairs (bp) longer than the native ENO1 promoter were isolated during the chemical synthesis. Gene expression of variant promoters was compared with that of the native promoter by measuring the amount of mRNA and the activity of beta-galactosidase by constructing ENO1-lacZ gene fusions. No significant differences were observed between the native and variant promoters in transcription levels. The start site of transcription was mapped on CAAG, a consensus sequence of transcription start site of yeast glycolytic genes. The results suggest a longer CT block in ENO1 promoter may not affect the expression of the yeast ENO1 gene. In addition, the level of ENO1 gene expression was found to be higher in stationary phase cells than in log phase cells.

The amino acids corresponding to the cleavage site of a hybrid preprotein containing a chicken lysozyme signal and a mature portion of human lysozyme were altered. The processing of mutant signals of -3Pro and -3Asp/-1Ala decreased remarkably, while that of -2Pro was 75% of that of the native signal. The major cleavage site of -3Pro was the same as that of the native signal, but that of the -2Pro and -3Asp/-I Ala signals was shifted one residue closer to the N-terminal side than the original site. The cleavage of the -2Pro signal, which was identical to the native processing of pheasant prelysozyme, suggested that the signal peptidases in yeast and bird are similar.

To investigate the structural requirements and functions of the N-terminal and the C-terminal regions of the chicken lysozyme signal peptide, the amino acids of each region were altered. The replacement of Gly(-1) and Leu(-2) with Pro(-1) and Ala(-2) or Val(-2), respectively, resulted in the complete shift of the cleavage site from position -1 to -2 in yeast (Saccharomyces cerevisiae). This shows that the introduction of a turn-promoting residue like Pro makes it possible to control the cleavage site of the signal peptide. Deletion of the positive charge and introduction of a negative charge in the N-terminal region decreased the lytic activity of secreted human lysozyme (HLY) and processing efficiency of preHLY, but the length and additional positive charge in this region had little influence. This suggests that the length of the N-terminal region scarcely influences the function of the signal peptide and that this region possibly interacts with the endoplasmic reticulum membrane to initiate the translocation of preprotein, similar to prokaryotic signal peptide. However, it needs only minimum positive charge for its function.