Figure 4.

Reinitiation is inhibited by a pseudoknot in the upORF that slows elongating ribosomes. The pseudoknot depicted in the boxed insert was positioned in the 5′ untranslated region (constructs 2 and 3), in the main coding domain (constructs 4 and 5) or in an upORF (construct 7). The polyacrylamide gels in (B) and (C) show protein yields from these structure-containing mRNAs and from unstructured control transcripts (constructs 1 and 8). The numeral preceding each mRNA in (A) matches the number of the lane in which that mRNA was tested (B and C). Construct 6 is the same as construct 5. Coding domains, which are not drawn to scale, are shown as filled boxes. The actual sequences of the mRNAs are given in Figure 3. Capped mRNAs were translated as usual at 25°C in reticulocyte lysate supplemented with [³H]leucine and 2 mM Mg²⁺.