Figure 5. Diphthamide modification on eEF2 promotes CrPV-IRES translation.
(A) Scheme for in vitro reconstituted translation elongation assay with CrPV-IRES RNAs. (B) Diagram of the secondary structure of the 200-nt CrPV-IRES. The coding region of the RNA was altered to encode the tripeptide MFK as indicated. Red asterisks denote the site of the CC to GG mutation in BP1 and BP2 of PKI. (C) Electrophoretic TLC analysis of peptide products from elongation assays programmed with the CrPV-IRES (left panel), the CC to GG mutant CrPV-IRES (second panel), or a model unstructured 52-nt mRNA encoding MFK (third panel). In the fourth panel, the same 52-nt model mRNA was assembled on 80S initiation complexes using the canonical translation initiation factors. The identities of spots corresponding to free methionine (M), dipeptide MF and tripeptide MFK are indicated. The fraction of MFK synthesis in the reactions with the WT CrPV-IRES and the 80S initiation complex were fit to a single exponential equation and plotted on the right. (D–E) Fraction of MFK synthesis in elongation assays programmed with 80S initiation complexes (D) or CrPV-IRES (E) and using purified WT eEF2, unmodified eEF2 lacking diphthamide (Unmod), eEF2-P580H, or eEF2-H699N, as indicated. Results were plotted and fit to a single exponential equation, and observed rate constants and end levels are shown (note the different time scales for the plots). Errors are standard deviation from three independent experiments.