title: Deletion of EFL1 results in heterogeneity of the 60 S GTPase-associated rRNA conformation.
abstract: Previous work suggested that the release of the nucleolar Tif6 from nascent 60 S subunits occurs in the cytoplasm and requires the cytoplasmic EF-2-like GTPase, Efl1. To check whether this release involves an rRNA structural rearrangement mediated by Efl1, we analyzed the rRNA conformation of the GTPase center of 80 S ribosomes in three contexts: wild-type, Deltaefl1 and a dominant suppressor R1 of Deltaefl1. This analysis was restricted to domain II and VI of 25 S rRNA. The rRNA analysis of R1 ribosomes allows us to distinguish the effects due to depletion of Efl1 from the resulting nucleolar deficit of Tif6. Efl1 inhibits the EF-2 GTPase activity, suggesting that the two proteins share a similar ribosome-binding site. The 80 S ribosomes from either type failed to show any difference of conformation in the two rRNA domains analyzed. However, the same analysis performed on the pool of free 60 S subunits reveals several rRNA conformational differences between wild-type and Deltaefl1 subunits, whereas that from the suppressor strain is similar to wild-type. This suggests that the nucleolar deficit of Tif6 during assembly of the 60 S preribosomes is responsible for the changes in rRNA conformation observed in Deltaefl1 60 S subunits. We also purified 60 S preribosomes from the three genetic contexts by TAP-tagging Tif6. The protein content of 60 S preribosomes associated with Tif6p in a Deltaefl1 strain are obtained at a lower yield but have, surprisingly, a protein composition that is a priori similar to that of wild-type and the suppressor strain.
authors.affiliation: UPR no. 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15, rue René Descartes, 67084 Strasbourg Cedex, France.
authors.forename: Jean-Sébastien
authors.initials: JS
authors.lastname: Graindorge
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Rousselle
authors.forename: Bruno
authors.initials: B
authors.lastname: Senger
authors.forename: Pascal
authors.initials: P
authors.lastname: Lenormand
authors.forename: Abdelkader
authors.initials: A
authors.lastname: Namane
authors.forename: François
authors.initials: F
authors.lastname: Lacroute
authors.forename: Franco
authors.initials: F
authors.lastname: Fasiolo
mesh_terms: D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002352:Carrier Proteins; D007381:Intermediate Filament Proteins; D010750:Phosphoproteins; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C491609:TIF6 protein, S cerevisiae; D020558:GTP Phosphohydrolases