Motif III in Superfamily 2 “Helicases” Helps Convert the Binding Energy of ATP into a High-Affinity RNA Binding Site in the Yeast DEAD-Box Protein Ded1 |
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Authors: | Josette Banroques Monique Doère Patrick Linder |
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Institution: | 1 Institut de Biologie Physico-chimique, CNRS UPR 9073 in association with the Université Paris VII, Paris 75005, France 2 Centre de Génétique Moléculaire, CNRS FRE 3144, Gif-sur-Yvette 91198, France 3 Département de Microbiologie et Médecine Moléculaire, Centre Médical Universitaire, Geneva 1211, Switzerland |
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Abstract: | Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a “helicase” strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (kcat), but not the affinity for ATP (Km). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of γ-PO4 of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the β,γ-phosphoanhydride bond of ATP. |
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Keywords: | SF2 superfamily 2 SF1 superfamily 1 ssRNA single-stranded RNA AMP-PNP adenosine 5&prime -(β γ-imino)triphosphate PDB Protein Data Bank SD-Leu plate synthetic minimal medium plate lacking leucine 5-FOA 5-fluoroorotic acid EMSA electrophoretic mobility shift assay |
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