Figure 4.
Toeprint analysis of ribosome stalling on poly-AAG/A and the UAA stop codon. (A and B) Schematic representation of reporters employed for toeprint analysis. The sequence of the transcript and translation product is shown in Supplementary Figure S4A. The TAA codon is shown in green. The position of the primer used for primer extension in toeprint experiments is indicated by the black arrow. (C and D) Toeprint analysis of the stalling reporter mGlk-K20-2HA-ss (C) and the readthrough reporter mGlk-stop-K20-2HA-ss (D). Translation reactions were performed in wild-type or Δzuo1 extract for 90 min at 20°C in the presence (+) or absence (−) of 300 μg/ml cycloheximide (CHX). If indicated purified RAC was added prior to the translation reaction as described in Figure 3. Primer extension was subsequently performed with ribosomal complexes collected by centrifugation as described in ‘Materials and Methods’ section. The control lane corresponds to a reaction without translation extract. Samples were separated on 7.5% urea-polyacrylamide gels together with the appropriate sequencing ladder (lanes 1–4). The full length extension product (FL) is indicated by the black arrow and the product corresponding to cycloheximide-stalled initiation complexes (16 nucleotides downstream of the AUG initiation codon) is indicated by the blue arrow (+16). The K20 sequence AAG-AAA-(AAG-AAG-AAA)6 is indicated by the pink ladder. The TAA stop codon of mGlk-stop-K20-2HA-ss is indicated in green. Colored arrows represent stops of the reverse transcriptase (toeprints) due to stalling of ribosomes on the K20 sequence (pink), or on the UAA stop codon (green). The position of the main toeprints are numbered relative to the first lysine codon of mGlk-K20-2HA-ss (C) and relative to the UAA stop codon of mGlk-stop-K20-2HA-ss (D). The black asterisks indicate toeprints due to ribosome stalling upstream of the K20 sequence. (E and F) Schematic representation of the toeprint results. Shown is the ribosome stalled with codon 7 (AAG) of the poly-AAG/A in the A-site (E) or with the UAA stop codon in the A-site (F). These ribosome stalls result in toeprints at +31 (relative to the first nucleotide of the first lysine codon) and position +13 (relative to the first nucleotide of the UAA), respectively.