title: The K⁺-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation.
abstract: Ribosome synthesis employs a number of energy-consuming enzymes in both eukaryotes and prokaryotes. One such enzyme is the conserved circularly permuted GTPase Nug1 (nucleostemin in human). Nug1 is essential for 60S subunit assembly and nuclear export, but its role and time of action during maturation remained unclear. Based on in vitro enzymatic assays using the Chaetomium thermophilum (Ct) orthologue, we show that Nug1 exhibits a low intrinsic GTPase activity that is stimulated by potassium ions, rendering Nug1 a cation-dependent GTPase. In vivo we observe 60S biogenesis defects upon depletion of yeast Nug1 or expression of a Nug1 nucleotide-binding mutant. Most prominently, the RNA helicase Dbp10 was lost from early pre-60S particles, which suggested a physical interaction that could be reconstituted in vitro using CtNug1 and CtDbp10. In vivo rRNA-protein crosslinking revealed that Nug1 and Dbp10 bind at proximal and partially overlapping sites on the 60S pre-ribosome, most prominently to H89 that will constitute part of the peptidyl transferase center (PTC). The binding sites of Dbp10 are the same as those identified for the prokaryotic helicase DbpA bound to the 50S subunit. We suggest that Dbp10 and DbpA are performing a conserved role during PTC formation in all organisms.
authors.affiliation: Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 328, Heidelberg D-69120, Germany.
authors.forename: Rizos-Georgios
authors.initials: RG
authors.lastname: Manikas
authors.affiliation: Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 328, Heidelberg D-69120, Germany.
authors.forename: Emma
authors.initials: E
authors.lastname: Thomson
authors.affiliation: Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 328, Heidelberg D-69120, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Thoms
authors.affiliation: Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 328, Heidelberg D-69120, Germany ed.hurt@bzh.uni-heidelberg.de.
authors.forename: Ed
authors.initials: E
authors.lastname: Hurt
mesh_terms: D001665:Binding Sites; D002597:Chaetomium / Q000235:genetics* / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics*; D020558:GTP Phosphohydrolases / Q000235:genetics* / Q000378:metabolism; D010458:Peptidyl Transferases / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000235:genetics*; D016601:RNA-Binding Proteins; D054683:Ribosome Subunits, Large, Eukaryotic / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D010458:Peptidyl Transferases; C411109:DBP10 protein, S cerevisiae; D020558:GTP Phosphohydrolases; C437988:Nug1 protein, S cerevisiae; D004265:DNA Helicases; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases
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