Figure 2. Specific protein contacts of nucleotides at different positions relative to the critical G nucleotide during Nsp1-mediated RNA cleavage.

(A) Sequences of 19nt.-long (CAA)n RNAs containing G14, dA at position −7 and 4thioU at indicated positions relative to G14. (B-K) UV cross-linking of 5'[³²P]-labeled 19nt.-long (CAA)n RNAs containing G14, dA at position −7 and 4thioU at indicated positions (panel A) with Nsp1, eIF3g and 40S subunits, assayed by SDS-PAGE and phosphor imaging. The positions of RNA and cross-linked Nsp1, eIF3g and ribosomal proteins are indicated. (L-M) UV cross-linking of 5'[³²P]-labeled 19nt.-long (CAA)n RNAs containing G14, dA at position −7 and 4thioU at positions −2 (L) or −1 (M) with wt and mutant Nsp1 in the presence of 40S subunits and eIF3g, assayed by SDS-PAGE and phosphor imaging. The positions of RNA and cross-linked Nsp1 and eIF3g are indicated. (N) Ribbon diagram of the Nsp1 N-terminal domain (PDB: 7K3N) to show mutated residues in stick colored depending on their importance for cross-linking of Nsp1 to positions −1 and −2 relative to the G nucleotide: red (crucial), orange (very strong) and green (moderate) sticks.