Figure 4. Model and probing of domain 3 in the ribosome P site.

(a) The part of CrPV IGR IRES domain 3 that mimics the tRNA anticodon loop (red) aligned with the structure of the anticodon loop of an authentic P site–bound tRNA^Met (gray, from PDB 2J00)²⁶. Dashed box, portion of the structure that is a precise mimic of the initiator tRNA anticodon loop. (b) View of the structure of CrPV domain 3 docked into its predicted position within the decoding groove, aligned with P site tRNA as in panel a. The CrPV domain 3 is in red and cyan, a portion of A site tRNA is in green, and P and E site tRNAs and mRNA are in gray. During CrPV IGR IRES-driven initiation, only the A site tRNA is present. (c) Native gel of mutants d3_6190–6191_comp (U6190A + A6191U) and d3_Δ6191 next to wild-type domain 3 in the presence of 10 mM MgCl₂. (d) A portion of an example gel of PSIV IGR IRES domain 3 modified with NMIA. Lanes containing free IRES RNA, 40S-bound IRES RNA and 80S-bound IRES RNA are shown over a dideoxy sequencing ladder. For clarity, this figure was constructed by splicing lanes from a single gel. Above the gel is a trace quantification of each lane. Some of the nucleotides with strong changes are labeled. (e) Graph of multiple SHAPE probing experiments quantified and analyzed. Gray dashed box, one s.d. between multiple measurements. The difference in modification between 40S-bound and 80S-bound IRES is shown, and changes greater than one s.d. are colored; red bars above the zero line, nucleotides that are modified more in the context of the 80S ribosome; blue bars under the line, those that are modified less. (f) Changes of panel e overlaid on the PSIV IGR IRES domain 3 secondary structure.