Figure 4.

ASCC Acts on Ribosome Queues to Facilitate Subunit Separation

(A) In vitro translation of endogenous mRNAs in RRL containing ³⁵S-methionine, 75 nM ZNF598, 0.8 μM eRF1^AAQ, and 50 nM ASCC, as indicated. Pactamycin was added after 10 min to inhibit additional initiation. Autoradiogram of the total reaction products, after digestion of tRNA with RNase, is shown. The positions of ~75 kDa lipoxygenase (Lox), ~15 kDa full-length (FL) globins, and truncated globins are indicated. The identity of two ³⁵S-labeled products (blue circles) is not known. They are specific to eRF1^AAQ-containing samples, co-migrate with polysomes (see C), partially diminished with ASCC, and too small to be ubiquitination.

(B and C) Reactions as in (A) were separated on sucrose gradients and analyzed under conditions where peptidyl-tRNA products are preserved. The region of the gel showing Lox is displayed in (B), and the region showing globins in the top part of (C). The positions of tRNA-attached polypeptides (verified by their shift upon RNase digestion), and full-length (FL) proteins are indicated. The bottom part of (C) shows the same samples analyzed after digestion with RNase A. The black and red arrows indicate full-length and truncated species of globins, respectively. Black asterisks indicate ³⁵S-methionyl-tRNA arising from translation-initiation complexes stabilized by pactamycin. Separate experiments (not shown) verified that this product is only seen with pactamycin and disappears with RNase digestion prior to electrophoresis. A minor product seen between the tRNA-attached and free Lox is probably the product of another endogenous mRNA.