6533b7d1fe1ef96bd125ce71

RESEARCH PRODUCT

TheSCH9 protein kinase mRNA contains a long 5′ leader with a small open reading frame

subject

description

The SCH9 yeast gene, that was previously identified as a suppressor of cdc25 and ras1- ras2-ts temperature-sensitive mutants, encodes a putative protein kinase that positively regulates the progression of yeast cells through the G1 phase of the cell cycle. We have determined the structure of the SCH9 transcription unit, using primer extension and S1 mapping techniques. The corresponding mRNA included an unusually long 5' region of more than 600 nucleotides preceding the major open reading frame (ORF). While the latter corresponded to a protein of 824 amino acids, an upstream open reading frame (uORF) within the 5' leader could potentially encode a 54 amino acid peptide. To investigate the role of the AUGs within the uORF, we engineered chimaeric plasmid vectors in which SCH9 sequences including the promoter, the mRNA leader and the first 514 nucleotides of the major ORF were fused in-frame with beta-galactosidase-coding sequences. Upon introduction into yeast cells, the fusion protein was efficiently expressed. However, mutational disruption of the uORF using oligonucleotide-directed mutagenesis did not affect the level of expression of the fusion protein. This indicates that regulatory mechanisms in Saccharomyces cerevisiae prevent upstream AUGs within the SCH9 mRNA leader sequence from influencing translation from downstream initiation codons.