Figure 2.

PTE-mediated cap-independent translation in mammalian systems. (A) Overlay of wheat (blue) and human (amber) eIF4E structures (Swiss PDB Viewer and PovRay, accession numbers 2IDV and 1IPC, respectively), showing tryptophan residues that sandwich around the m⁷GTP cap. (B) Cis-translation results of uncapped viral reporter RNAs (CITE constructs, Figure 1A) in rabbit reticulocyte lysate (RRL), with relative light units (RLU) shown relative to those produced by the CarMV CITE construct. BlucB is a luciferase reporter containing the 5′ and 3′ UTRs of barley yellow dwarf virus (BYDV), which harbors a BYDV-like translation element (BTE) rather than a PTE in its 3′ UTR. (C) Trans-inhibition of translation of uncapped PEMV2 CITE construct mRNAs by PTEs as in Figure 1C, but in RRL. (D) Cis-translation of uncapped viral CITE construct RNAs in HeLa cell lysate. RLUs are relative to those obtained from firefly luciferase RNA containing the EMCV IRES in its 5′ UTR. (E) Cis-translation results of viral CITE construct RNAs relative to EMCV reporter in DNA-transfected BHK-T7 cells. Plasmids were linearized with Sma I as for in vitro transcription of RNAs used in in vitro assays. The data shown are averages of triplicates from at least two experiments, with error bars representing standard error.