Figure 1.

Nascent Chain-tRNAs on Stalled Ribosomes Are Ubiquitinated In Vitro

(A) Line diagram of truncated and full-length (FL) β-VHP transcripts.

(B) Schematic diagram of a stalled β-VHP ribosome-nascent chain complex (RNC).

(C) In vitro translation reactions of β-VHP RNCs containing FLAG-tagged ubiquitin were sequentially treated with RNase, precipitated with CTAB, and/or immunoprecipitated with anti-FLAG or control antibodies as indicated. The positions of the nascent chain (NC) containing or lacking a tRNA are indicated, along with ubiquitinated products (Ub).

(D and E) Translation reactions of β-VHP or β-VHP-FL containing His-tagged ubiquitin were separated on 10%–50% sucrose gradients. Each fraction was immunoprecipitated with an antibody against the substrate and visualized by autoradiography. The upper portion of the gel was exposed 4-fold longer than the lower portion to visualize ubiquitinated products. An A₂₆₀ trace is shown to indicate ribosomal fractions. An aliquot of pooled fractions 5–7 was also subject to immobilized Co²⁺ pull-downs to recover His-tagged ubiquitinated products (last lane). See also Figure S1.