MUTATIONAL ANALYSIS ON THE RNA HELICASE DOMAIN OF THE YEAST ROK1 GENE

The ROK1 gene was previously identified as a high-copy number plasmid suppressor of the kem1 null mutation that affects microtubule and spin dle pole body functions in Saccharomyces cerevisiae. The Rok1 protein sequence contains highly conserved domains found in the DEAD protein f amily of ATP-dependent RNA helicase. To investigate the crucial roles of the RNA helicase domains found in the yeast ROK1 gene, we have intr oduced amino acid substitution mutations in these regions. Two mutatio ns were identified in the DEAD domain, which substitute the first Asp with Glu. The D280E1 mutation, when subcloned onto the high-copy yeast plasmid YEp24, no longer suppresses the slow growth phenotype and the benomyl hypersensitivity of the kem1 mutation. Furthermore, the same mutation does not complement the lethality caused by the rok1 null mut ation. These results suggest that the DEAD helicase domain is crucial to the ROK1 function.