Fig. 1: In vitro reconstitution of the ubiquitinated colliding ribosomes.
From: Decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex
a Schematic drawing of the SDD1 model mRNA used in the in vitro translation (IVT) assay (Top). Schematic of the in vitro experiments (bottom). b, c The purified RNCs in the IVT reaction using HEL2-containing or hel2-knockout (hel2∆) IVT extract were separated by sucrose density gradient centrifugation and detected by UV absorbance at a wavelength of 260 nm. HA-tagged uS10 in each fraction was detected by immunoblotting using an anti-HA antibody. Immunoblotting of purified RNCs using anti-HA (d), K63-linkage (e), and K48-linkage-specific anti-ubiquitin antibodies (f). g In vitro ubiquitination assay. Purified RNCs derived from the hel2-knockout (hel2∆) IVT reaction mixed with Hel2 (E3), Uba1 (E1), Ubc4 (E2), ATP, and ubiquitin or the indicated ubiquitin mutants. After the reaction, HA-tagged uS10 was detected by immunoblotting using an anti-HA antibody. All experiments were performed at least twice with highly reproducible results.
