A promoter activity in Saccharomyces cerevisiae of the 3′-noncoding region of the basidiomycetous mushroom gene

Abstract

The 3′-noncoding region of the priB gene derived from the basidiomycete Lentinus edodes was found to contain one GC box-like sequence, two CAAT boxes, two TATA box-like sequences and two pyrimidine-rich stretches (CT-motifs). It also contained a 16-bp sequence similar to the consensus sequence for PRIB protein binding and a short (61 bp) intron. Single-strand-specific S1 nuclease analysis of plasmid pBR322 DNA containing the 3′-noncoding region propagated in Escherichia coli revealed that this region forms an unusual, extended open structure within/around the downstream pyrimidine/purine (CT/AG)-biased sequence. To examine the promoter activity of the priB 3′-noncoding region in Saccharomyces cerevisiae, in which an autonomously replicating plasmid vector is available, we constructed two plasmids, YEp3′NCR-lacZ and YEp3′UTR-lacZ. The former contains the priB 3′-noncoding region and the reporter E. coli β-galactosidase gene while the latter contains the priB 3′-noncoding region lacking the intron and the reporter gene. The yeast transformants; obtained through introductions of these plasmids showed β-galactosidase activity and the activity conferred by YEp3′NCR-lacZ was about 50% of that conferred by YEp3′UTR-lacZ. The primer extension analysis showed that transcription of the reporter gene on both plasmids starts at three alternative sites all of which are located within the downstream CT-motif, suggesting a role for the unusual structure of the CT/AG-biased sequence in the initiation of transcription.