Figure 3

Translation of the Tat RNA in the RRL system. A. Structure of the recombinant Glob- and EMCV RNA templates. All recombinant RNAs encode LacZ as the sole gene for the monocistronic RNAs, and as the 3' one for the bicistronic RNAs. The 5' UTR sequences correspond to either the complete 5' leader of the globin mRNA or the EMCV leader (see materials and methods). B. Translation of the mono- and bicistronic RNAs in the RRL system. RNAs were 5' capped (+) or not (-). Note that the non-capped mono-Glob RNA was translated at 15% (lane 3) of the control level (lane 2) while the non-capped mono-EMCV RNA was translated at 125% (lane 6) of the control level (lane 5). LacZ was not translated with the Bi-Glob RNA (lane 4) while it was at 90% (lane 7) the control level with the Bi-EMCV RNA. C. Structure of the recombinant Tat RNAs. The monocistronic and bicistronic recombinant Tat RNAs are shown. D. Translation of the mono- and bicistronic Tat RNAs in the RRL system. Note that mono-Tat1 and mono-Tat2 were translated at the same level either capped (lanes 1 and 4) or non-capped (lanes 2 and 5). Translation of the Tat ORF occurred with the Bi-Tat RNAs, but levels were about 40% (lanes 3 and 6) of the control levels (lanes 1 and 5). E. Translation of the Tat RNAs in the presence of the cap analog 7m-Gppp. Translation conditions were as in B (upper panel), but contained 7m-Gppp (lower panel) during the whole reaction (see methods). Translation of the mono-Glob RNA was extensively inhibited (lane 2) but this was not seen with mono-EMCV RNA (lanes 4 and 5), as expected. Note that translation of the Tat RNAs was not inhibited by the cap analog (compare lanes 6-9 in upper and lower panels).