ATP hydrolysis activity of the DEAD box protein Rok1p is required for in vivo ROK1 function

The yeast ROK1 gene has been initially identified as a high copy plasmid su ppressor of the kem1 null mutation and implicated in microtubule-mediated f unctions. Based on the deduced amino acid sequence of the ROK1 gene, Rok1p has been classified in the DEAD protein family of ATP-dependent RNA helicas es, A subsequent report has suggested that Rok1p is required for rRNA proce ssing. We report here the first study on the biochemical activity associate d with Rok1p, The MBP-Rok1 hybrid protein was synthesized in Escherichia co il and purified by amylose affinity column and ion exchange chromatography. Rok1p has ATP hydrolysis activity. The significance of the conserved ATPas e domains was addressed by generating a series of amino acid substitution m utations in these domains. Both in vivo lethality tests of the mutations an d biochemical characterization of the mutant proteins suggest that ATP hydr olysis activity of Rok1p is essential for ROK1 function. The ATPase activit y of Rok1p appears to be independent of single-stranded RNA. Furthermore, r eplacement of the first Arg in the HRIGR domain, the known RNA-binding doma in, with Thr, Ile or Lys has no detectable effect on in vivo ROK1 function. The lack of RNA dependency and some of the mutational phenotypes of ROK1 d ifferentiate this gene from other members of the family.