Nucleic Acids Research, Vol 27, Issue 13 2753-2759, Copyright © 1999 by Oxford University Press
JY Oh and J Kim
The yeast ROK1 gene has been initially identified as a high copy plasmid
suppressor of the kem1 null mutation and implicated in microtubule-mediated
functions. Based on the deduced amino acid sequence of the ROK1 gene, Rok1p
has been classified in the DEAD protein family of ATP-dependent RNA
helicases. A subsequent report has suggested that Rok1p is required for
rRNA processing. We report here the first study on the biochemical activity
associated with Rok1p. The MBP-Rok1 hybrid protein was synthesized in
Escherichia coli and purified by amylose affinity column and ion exchange
chromatography. Rok1p has ATP hydrolysis activity. The significance of the
conserved ATPase domains was addressed by generating a series of amino acid
substitution mutations in these domains. Both in vivo lethality tests of
the mutations and biochemical characterization of the mutant proteins
suggest that ATP hydrolysis activity of Rok1p is essential for ROK1
function. The ATPase activity of Rok1p appears to be independent of
single-stranded RNA. Furthermore, replacement of the first Arg in the HRIGR
domain, the known RNA-binding domain, 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 differentiate this gene from
other members of the family.
ARTICLES
ATP hydrolysis activity of the DEAD box protein Rok1p is required for in vivo ROK1 function
Department of Microbiology, College of Natural Sciences, Chungnam National University, Taejeon 305-764, Korea.
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