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. 2021 Mar 17;13(3):493. doi: 10.3390/v13030493

Figure 4.

Figure 4

Toeprinting analysis of 80S-aNCV IRES complexes. (A) Schematic of IGR IRES PKI region. Mutations within the PKI region are annotated. (B) Primer extension analysis was performed on in vitro transcribed bicistronic RNAs containing the indicated wild-type or mutant CrPV IRES (left) or aNCV IRES or CrPV IRES (right) incubated in rabbit reticulocyte lysate (RRL) pretreated with cycloheximide (0.68 mg/mL). For the aNCV IRES, the sequencing ladder corresponds to nucleotides 279–331. The major toeprint is indicated at right, which is twenty nucleotides downstream of AUC289-91 of PKI given that the A is +1. (C) Primer extension analysis on in vitro transcribed bicistronic RNAs containing the indicated wild-type or mutant aNCV IRES incubated with purified ribosomes. Sequencing ladder corresponds to aNCV IRES nucleotides 269–319. The major toeprint is indicated at right, which is 13 nucleotides downstream of AUC289-91. Shown is a representative gel from at least three independent experiments.