Fig. 4: Rpf2–Rrs1 C-terminal extensions are required for 5S RNP assembly into pre-60S ribosomes.
From: Structure of nascent 5S RNPs at the crossroad between ribosome assembly and MDM2–p53 pathways
a, SDS–PAGE analysis of the yeast 5S RNP complexes, Syo1 lacking (top) or Syo1 containing (bottom), with either wild-type (WT) or truncated (ΔC/ΔC) Rpf2/Rrs1 factors, used for the EMSA. Asterisks indicate bait proteins used for the split-tag affinity purification. The purifications of these 5S RNP complexes were done more than three times with consistent results. b, Folding prediction, calculated by RNAfold46, of the yeast 25S rRNA fragment H81–H87 (red, right) and tRNAPhe (blue, left) used for the EMSA with the 5S RNP complexes. The colored areas illustrate the contacts of the 5S RNP proteins to the 25S rRNA helixes (labeled from H81 to H87) after binding to pre-60S ribosome (PDB ID 3JCT). c, EMSA radiographs showing the specific band shift (asterisks) of the radiolabeled 25S rRNA fragment upon binding to increasing amounts of the indicated 5S RNP complexes. nt, nucleotides. All of the EMSA assays were done twice with a similar outcome. d, SDS–PAGE analysis of sucrose gradient fractions from in vivo 5S RNP assembly into pre-60S ribosomes in yeast cells. 5S RNP incorporation into pre-60S particles was monitored upon affinity purification using Rpf2 as bait, from yeast wild-type, rpf2∆C and rrs1∆C single mutants, and rpf2∆C rrs1∆C double mutants. The typical protein pattern of pre-60S ribosomes is visible in the fractions with high molecular weight (lane 5), whereas the free 5S RNP (unbound) is visible in the fractions with low molecular weight (lanes 1 and 2). The bands corresponding to the 5S RNP factors were identified by mass spectrometry and are labeled. Co-precipitation of Fpr3 (triangle) and Fpr4 (square) with the free 5S RNP was also detected. This in vivo binding experiment was done at least twice with a similar outcome.
