Figure 2.

Site-Specific Stalling of Native Polysomes Triggers ZNF598 Engagement

(A) Strategy to site-specifically stall ribosomes at the stop codon on native polysomes translating globin mRNA using the mutant release factor eRF1^AAQ.

(B) The polysomes in native reticulocyte lysate (RRL) were allowed to elongate in a translation reaction supplemented with 1 μM eRF1^AAQ, 5 nM FLAG-tagged ZNF598, and 10 μM His-tagged ubiquitin. After fractionation on a sucrose gradient, translated globin polypeptides were detected by autoradiography and the other products by immunoblotting. Ubiquitinated eS10 was detected with anti-eS10 after pull-down via the His-tag on ubiquitin. FL indicates full-length nascent chains; “trunc. NCs” are truncated nascent chains.

(C) Native RRL polysomes elongated in the absence or presence of 1 μM eRF1^AAQ were incubated without or with micrococcal nuclease, ubiquitinated with ZNF598, and analyzed for eS10 by immunoblotting.

(D) Native RRL polysomes stalled with eRF1^AAQ were fractionated by a high resolution sucrose gradient (see Figure S2B). Fractions enriched in mono-, di-, tri-, tetra-, and penta-ribosomes were normalized to contain an equal number of ribosomes, ubiquitinated with 10 nM ZNF598, and analyzed by immunoblotting for eS10. The stained blot verifies equal input ribosomes in each reaction.

(E) Reactions corresponding to the third and fourth lanes of (C) were fractionated on a sucrose gradient and analyzed by Coomassie staining or immunoblotting. The positions of mono- and poly-ribosomes are indicated. The primary bands seen in the stained gel are ribosomal proteins.

See also Figure S2.