Figure 5. The mechanism and factor requirements for initiation on genomic and subgenomic SARS-Cov-2 mRNAs.
(A) Secondary structure model of the SARS-CoV-2 genomic 5’UTR annotated to show the ORF1 viral initiation codon AUG266, as well as CUG59 and AUG107 codons with their corresponding short upstream ORFs uORF1a and uORF1b. Mutations (red) introduced into SL1, SL2 and SL3 are shown in boxes. (B, C) Factor requirements for 48S complex formation on genomic (B) and subgenomic (C) SARS-CoV-2 mRNAs assayed by toe-printing. The positions of 48S complexes formed on different codons are indicated on the right. (D) The influence of destabilization of SL4 on 48S complex formation on CUG59. Upper panel - nucleotide substitutions (red) shown on secondary structure models of wt and destablized SL4. Lower panel - 48S complex formation assayed by toe-printing. The position of CUG59 is indicated on the left. (B-D) Lanes C, T, A, and G show the corresponding sequences derived using the same primers as for toe-printing. (E) The effect of mutations introduced into the SARS-CoV-2 5’UTR (some shown in panel A) on its activity, assayed by translation in RRL in the presence of 35S-Methionine. (F) The influence of RRL supplementation with eIF1 and eIF1A on initiation at CUG59 and AUG107 of SARS-CoV-2 mRNA lacking stop codons UAA134–6 and UAA137–9. (E-F) Initiation codons corresponding to translation products are indicated on the sides.