Extended Data Fig. 6: Yeast 5S RNP in vitro binding reconstitution into pre-60S particles using epitope-labeled subunits.
From: Structure of nascent 5S RNPs at the crossroad between ribosome assembly and MDM2–p53 pathways
a–c, The yeast hexameric 5S RNP was incubated with Nsa1-FtpA pre-60S particles, either non-depleted (a) or 5S RNP-depleted via GAL::HA-RPF2 (b), and as a further control, with Yvh1-FtpA-derived pre-60S particles (c). After sucrose gradient centrifugation of the assay mixtures, all fractions (fractions 1–6) were analyzed by SDS-PAGE (upper panel), and western blotting using the anti-HA antibody (middle panel). The bands corresponding to HA-Rpf2, uL18-HA, FLAG-HA-Rrs1, and uL5-HA are indicated. The different Nsa1-FtpA and Yvh1-FtpA pre-60S particles without addition of 5S RNP were also analyzed by sucrose gradient centrifugation (lower panels). M: Molecular weight marker. This in vitro binding assay has been done at least twice with a similar outcome.
