Folding a stable RNA pseudoknot through rearrangement of two hairpin structures

Folding messenger RNA into specific structures is a common regulatory mechanism involved in translation. In Escherichia coli, the operator of the rpsO gene transcript folds into a pseudoknot or double-hairpin conformation. S15, the gene product, binds only to the pseudoknot, thereby repressing its own synthesis when it is present in excess in the cell. The two RNA conformations have been proposed to exist in equilibrium. However, it remained unclear how structural changes can be achieved between these two topologically distinct conformations. We used optical tweezers to study the structural dynamics and rearrangements of the rpsO operator RNA at the single-molecule level. We discovered that the two RNA structures can be interchanged spontaneously and the pseudoknot can exist in conformations that exhibit various levels of stability. Conversion from the double hairpin to a pseudoknot through potential hairpin–hairpin interactions favoured the high-stability conformation. By contrast, mutations that blocked the formation of a hairpin typically resulted in alternative low-stability pseudoknots. These results demonstrate that specific tertiary interactions of RNA can be established and modulated based on the interactions and rearrangements between secondary structural components. Our findings provide new insight into the RNA folding pathway that leads to a regulatory conformation for target protein binding.