Recoveries of chimeric RNAs after incubation in Drosophila embryo extract. Forward or flipped constructs containing the SRE+ or SRE− regulatory RNAs depicted in Figure 3 were incubated in embryo extract for 30 min at 25°C. One sample was oligo(dT)-purified immediately (0 min), a second sample at the end of the incubation. RNAs were analyzed by denaturing gel electrophoresis (see Materials and Methods). The first lane contains a mixture of nLuc and a regulatory RNA as markers. Recoveries of the nLuc and regulatory RNAs are given at the bottom. RNA recovered at the 0 min time point was set to 100%. As recovery on oligo(dT) beads was based on the poly(A) stretch in the regulatory RNA, copurification of the nLuc RNA demonstrated the stability of chimeras. Furthermore, SRE+-containing constructs were more stable than SRE− constructs; thus, the lower translation yield of SRE+ RNAs was not due to RNA destabilization. Recoveries of regulatory RNAs were generally higher, presumably due to their small size (compare the small regulatory RNAs in the first two sets to the long fLuc-containing regulatory RNA in the third set). Recall that not all regulatory RNAs were bound to nLuc RNA (compare Fig. 2B).
