In Vitro Translation Assays for Ribosome-Associated mRNA

All transcripts utilized PCR products as templates for in vitro transcription (Sharma et al., 2010 (link)) except β-VHP-SL, which was transcribed from linearized plasmid. β-VHP-pA transcript was generated by treating β-VHP transcript with poly(A) polymerase to add an ∼200 nt poly(A) tail. ³³P-labeled transcripts were generated with ³³P-UTP (Perkin-Elmer) in transcription reactions. In vitro translation reactions using rabbit reticulocyte lysate (RRL), phenyl-depleted RRL, and DEAE fractionated RRL (Fr-RRL) were done as before (Sharma et al., 2010 (link); Hessa et al., 2011 (link)). ΔHbs1 RRL was generated by incubating 800 μl RRL with 200 μl of Pelota resin (immobilized via CnBr). Unconjugated and quenched CnBr resin served as a control. Unless indicated otherwise, translation reactions were for 60 min at 32°C. Where indicated, WT or DN Hbs1 was added at 10 min together with 100 μM aurin tricarboxylic acid to inhibit initiation. For direct analyses, translation reactions were denatured in 1% SDS and heated to 100°C. For downstream applications, translation reactions were cooled on ice and manipulated at 0°C–4°C for RNC isolation, sucrose gradients, and native immunoprecipitations (IPs).