Toeprinting analyses of mouse MOR containing uORFs that regulate ribosome leaky scanning

Abstract

<p><b>Copyright information:</b></p><p>Taken from "Translational repression of mouse mu opioid receptor expression via leaky scanning"</p><p></p><p>Nucleic Acids Research 2007;35(5):1501-1513.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865057.</p><p>© 2007 The Author(s).</p> Synthetic RNA (100 ng) transcripts were used to program translation mixtures derived from RRL. Mouse MOR-LUC RNA containing the wild-type uORFs [uAUG(+)] or deleted of all three uORFs [uAUG(−)] were incubated at 30°C for 15 min in reaction mixtures without cycloheximide (None) or supplemented with cycloheximide (Cyh) either prior to incubation (T0) or after 5 min of incubation at 30°C (T5). Control samples of RNA in reaction mixtures without extract (-EXT) or reaction mixtures containing extract without mRNA (-RNA) are indicated. The positions of the cDNA extension products corresponding to the mRNA 5′ end, uORF initiation (open triangles, asterisks) and LUC initiation codon (filled triangle) by radiolabeled mToe2 primer are indicated. During leaky scanning, ribosomes scan and load at either the upstream or downstream start codons. Adding cycloheximide at T0 traps ribosomes at the positions where they first load on the mRNA. Adding it during steady-state translation (T5) traps ribosomes where they are loaded, following the primary initiation event and subsequent reinitiation events