Deletion of EFL1 results in heterogeneity of the 60 S GTPase-associated rRNA conformation.
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.
Graindorge
Jean-Sébastien
JS
UPR no. 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15, rue René Descartes, 67084 Strasbourg Cedex, France.
Rousselle
Jean-Claude
JC
Senger
Bruno
B
Lenormand
Pascal
P
Namane
Abdelkader
A
Lacroute
François
F
Fasiolo
Franco
F
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
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