Ribosome toe printing assay

Toe printing assay was adapted from previously established protocols (Martin et al., 2016; Martin et al., 2011). Briefly, RRLs were incubated for 5 min at 30°C then 10 min on ice with buffer containing 1 U/μl of recombinant RNasin (Promega), 75 mM KCl 0.5 mM MgCl₂, and 1.3 mM of puromycin prior to initiation complex assembly. Then, the pre-initiation complexes were formed by incubation with 500 nM of the RNA of interest in the presence of specific inhibitors such as cycloheximide (1 mg/ml) or GMP-PNP (4 mM) for 5 min at 30°C and then 20 min on ice. Then, the pre-initiation complexes were complemented with one volume of ice-cold buffer A containing 20 mM Tris-HCl (pH 7.5), 100 mM KAc, 2.5 mM MgAc₂, 2 mM DTT, 1 mM ATP, and 0.25 mM spermidine and placed on ice. In order to separate ribosomal complexes from the non-ribosomal fraction, samples were centrifuged at 88,000 rpm in S100AT3 rotor (Sorvall-Hitachi) at 4°C for 1 hr. After centrifugation, the pellets containing the pre-initiation complexes were resuspended in 30 μl of ice-cold buffer A and incubated with 5’ radioactively labelled DNA oligonucleotide complementary to nts 22–51 of RLuc coding sequence for 3 min at 30°C. Then, 1 μl of a 320 mM Mg(Ac)₂, 4 μl of a dNTP mixture (containing 5 mM of dATP, dGTP, dTTP, and dCTP), 10 units of recombinant RNAsin (Promega), and 1 unit/μl AMV reverse transcriptase (Promega) were added and incubated for 1 hr at 30°C. The synthesized cDNAs were analysed on 8% PAGE next to sequencing ladders.