FIG. 3.
For in vitro translation of BPV L1 sequences, wt BPV L1 (wt) or synthetic L1 (HB) sequences were translated in vitro, using RRL or wheat germ extract in the presence of [35S]methionine. (A) wt L1 or HB L1 plasmid DNA was added to the T7-DNA polymerase coupled in vitro translation system (Promega), and L1 protein was translated for 20 min at 30°C. L1 protein was detected by PAGE and autoradiography. The BPV L1 wt gene produced a weak L1 band at 55 kDa (lane 1). The synthetic HBL1 gene made a larger amount of L1 protein (lane 2). The translation reaction where only vector was added produced no visible band (lane 3). When 10−5 to 10−9 M (lanes 4 to 8) bovine liver aminoacyl-tRNA was added to the translation system, the protein translation efficiency from wt L1 sequence was improved in a tRNA dose-dependent manner. (B) Comparison of the translation efficiencies of wt L1 and HB L1 sequences in the presence and absence of tRNA. Translation was carried out in RRL (rabbit) or wheat germ extract (wheat), and samples were collected every 2 min starting from min 8. Addition of 10−5 M bovine liver or yeast tRNA is indicated at the left.