Extended Data Fig. 4: In vitro reconstitution of Vms1 peptidyl-tRNA hydrolase activity. | Nature

Extended Data Fig. 4: In vitro reconstitution of Vms1 peptidyl-tRNA hydrolase activity.

From: Vms1 and ANKZF1 peptidyl-tRNA hydrolases release nascent chains from stalled ribosomes

Extended Data Fig. 4

a, Coomassie blue-stained gels of the indicated purified proteins used for in vitro reconstitution. b, Analysis of ribosome–nascent chain complexes generated by translation in reticulocyte lysate. PCR products encoding Flag–CRPNS followed by a 30-nucleotide poly(A) sequence and Flag–CRPStop (with a stop codon) were transcribed and translated in reticulocyte lysate in the presence of 35S-labelled methionine. Completed translation reactions were treated (or not treated), fractionated using SDS–PAGE, and visualized by autoradiography. Lane one, no treatment; lane two, RNase A treatment; lane three, CTAB treatment; lane four, immunoprecipitated with anti-Flag resin. For lanes three and four, the pellet fraction was analysed. c, Sucrose gradient analysis of reticulocyte translation reactions. Flag–CRPNSKn was transcribed and translated in 200 μl reticulocyte lysate for 30 min. Lysates were layered onto 2-ml 10–50% sucrose gradients and centrifuged using a Beckman SW55 rotor for 80 min. Eleven fractions (200 μl each) were collected from the top by hand. Aliquots were analysed for fractionation of 35S-labelled substrate and Rpl8 by autoradiography and immunoblotting, respectively. d, Quantification of two independent biological replicates of the yeast His6–Vms1 and His6–Vms1-Q295L titration reactions shown in Fig. 4d. e, Quantification of the human ANKZF1 and ANKZF1-Q246L titration reactions shown in Fig. 4e. Data are representative of two biological replicates. Gel source images are provided in Supplementary Fig. 1 and Source Data are available with the online version of this paper.

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