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Characterization of the ATPase and unwinding activities of the yeast DEAD-box protein Has1p and the analysis of the roles of the conserved motifs

Rocak, Sanda ; Emery, Bertrand ; Tanner, N. Kyle ; Linder, Patrick

In: Nucleic Acids Research, 2005, vol. 33, no. 3, p. 999-1009

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    Title
    • Characterization of the ATPase and unwinding activities of the yeast DEAD-box protein Has1p and the analysis of the roles of the conserved motifs
    Author
    • Rocak, Sanda. Departement de Microbiologie et Médecine Moléculaire, Centre Médical Universitaire 1, rue Michel Servet, CH-1211 Geneve 4, Switzerland
    • Emery, Bertrand. Departement de Microbiologie et Médecine Moléculaire, Centre Médical Universitaire 1, rue Michel Servet, CH-1211 Geneve 4, Switzerland
    • Tanner, N. Kyle. Departement de Microbiologie et Médecine Moléculaire, Centre Médical Universitaire 1, rue Michel Servet, CH-1211 Geneve 4, Switzerland
    • Linder, Patrick. Departement de Microbiologie et Médecine Moléculaire, Centre Médical Universitaire 1, rue Michel Servet, CH-1211 Geneve 4, Switzerland
    Document Type
    • Postprint
    Language
    • English
    Published in
    • Nucleic Acids Research, 2005, vol. 33, no. 3, p. 999-1009. Oxford University Press
    Other Version
    Classification
    • Health
    OAI-PMH Identifier
    • oai:doc.rero.ch:290956
    Summary
    The yeast DEAD-box protein Has1p is required for the maturation of 18S rRNA, the biogenesis of 40S r-subunits and for the processing of 27S pre-rRNAs during 60S r-subunit biogenesis. We purified recombinant Has1p and characterized its biochemical activities. We show that Has1p is an RNA-dependent ATPase in vitro and that it is able to unwind RNA/DNA duplexes in an ATP-dependent manner. We also report a mutational analysis of the conserved residues in motif I (86AKTGSGKT93), motif III (228SAT230) and motif VI (375HRVGRTARG383). The in vivo lethal K92A substitution in motif I abolishes ATPase activity in vitro. The mutations S228A and T230A partially dissociate ATPase and helicase activities, and they have cold-sensitive and lethal growth phenotypes, respectively. The H375E substitution in motif VI significantly decreased helicase but not ATPase activity and was lethal in vivo. These results suggest that both ATPase and unwinding activities are required in vivo. Has1p possesses a Walker A-like motif downstream of motif VI (383GTKGKGKS390). K389A substitution in this motif significantly increases the Has1p activity in vitro, which indicates it potentially plays a role as a negative regulator. Finally, rRNAs and poly(A) RNA serve as the best stimulators of the ATPase activity of Has1p among the tested RNAs