FIGURE 2.

The 5′ UTR of gypsy promotes translation in the context of mono- and dicistronic RNAs. (A) The complete 5′ UTR (846 nt) of gypsy was inserted upstream of the lacZ reporter gene (pMC-WT construct). Several concentrations of uncapped (lane 1, 5μg/mL; 2, 10 μg/mL; 3, 20 μg/mL; 4, 40 μg/mL) and capped RNAs (lane 5, 5μg/mL; 6, 10 μg/mL; 7, 20 μg/mL; 8, 40 μg/mL) were translated for 60 min in the RRL (10 μL). (B) The 5′ UTR was inserted between the two cistrons of a bicistronic construct coding for neomycin and β-Gal proteins (pDC-WT). Several concentrations of capped (lane 2, 10 μg/mL; 3, 20 μg/mL; 4, 40 μg/mL) and uncapped (lane 5, 10 μg/mL; 6, 20 μg/mL; 7, 40 μg/mL) bicistronic RNAs were translated as described above. A control assay with 10 μg/mL of capped bicistronic EMCV-D260-837 (DC-EMCV) was set in parallel (lane 1). At the end of a 60-min incubation, 1 μL sample of each assay was analyzed on a 15% SDS-PAGE and subjected to autoradiography. Positions of molecular weight markers (kD), neomycin (Neo), and β-Gal translation products are indicated. Note that β-Gal synthesized from the EMCV construct migrates slightly faster on SDS-PAGE (lane 1), which is due to linearization of the DNA at a different site (see Materials and Methods). This difference in size does not alter the methionine content of the protein.