  Prediction of the functions of riboswitches requires a quantitative
description of the folding landscape so that the barriers and time scales for
the conformational change in the switching region in the aptamer can be
estimated. Using a combination of all atom molecular dynamics and
coarse-grained model simulations we studied the response of adenine (A) binding
add and pbuE A-riboswitches to mechanical force. The two riboswitches contain a
structurally similar three-way junction formed by three paired helices, P1, P2,
and P3, but carry out different functions. Using pulling simulations, with
structures generated in MD simulations, we show that after P1 rips the dominant
unfolding pathway in add A-riboswitch is the rupture of P2 followed by
unraveling of P3. In the pbuE A-riboswitch, after P1 unfolds P3 ruptures ahead
of P2. The order of unfolding of the helices, which is in accord with single
molecule pulling experiments, is determined by the relative stabilities of the
individual helices. Our results show that the stability of isolated helices
determines the order of assembly and response to force in these non-coding
regions. We use the simulated free energy profile for pbuE A-riboswitch to
estimate the time scale for allosteric switching, which shows that this
riboswitch is under kinetic control lending additional support to the
conclusion based on single molecule pulling experiments. A consequence of the
stability hypothesis is that a single point mutation (U28C) in the P2 helix of
the add A-riboswitch, which increases the stability of P2, would make the
folding landscapes of the two riboswitches similar. This prediction can be
tested in single molecule pulling experiments.
