Translational control by delayed RNA folding: Identification of the kinetic trap

The maturation or A-protein gene of single-stranded RNA phage MS2 is preced ed by a 130-nt long untranslated leader. When MS2 RNA folding is at equilib rium, the gene is untranslatable because the leader adopts a well-defined c loverleaf structure in which the Shine-Dalgarno (SD) sequence of the matura tion gene is taken up in long-distance base pairing with an upstream comple mentary sequence (UCS), Synthesis of the A-protein takes place transiently while the RNA is synthesized from the minus strand, This requires that form ation of the inhibitory cloverleaf is slow, In vitro, the folding delay was on the order of minutes. Here, we present evidence that this postponed fol ding is caused by the formation of a metastable intermediate. This intermed iate is a small local hairpin that contains the UCS in its loop, thereby pr eventing or slowing down its pairing with the SD sequence, Mutants in which the small hairpin could not be formed made no detectable amounts of A-prot ein and were barely viable. Apparently, here the cloverleaf formed quicker than ribosomes could bind. On the other hand, mutants in which the small in termediary hairpin was stabilized produced more A-protein than wild type an d were viable, One hardly growing mutant that could not form the metastable hairpin and did not make detectable amounts of A-protein was evolved. The emerging pseudo-revertant had selected two second site repressor mutations that allowed reconstruction of a variant of the metastable intermediate. Th e pseudo-revertant had also regained the capacity to produce the A-protein.