posted on 2021-12-27, 16:34authored byAndrea Marton Menendez, David J. Nesbitt
Riboswitches play an important role
in RNA-based sensing/gene regulation
control for many bacteria. In particular, the accessibility of multiple
conformational states at physiological temperatures allows riboswitches
to selectively bind a cognate ligand in the aptamer domain, which
triggers secondary structural changes in the expression platform,
and thereby “switching” between on or off transcriptional
or translational states for the downstream RNA. The present work exploits
temperature-controlled, single-molecule total internal reflection
fluorescence (TIRF) microscopy to study the thermodynamic landscape
of such ligand binding/folding processes, specifically for the Bacillus subtilis lysine riboswitch. The results
confirm that the riboswitch folds via an induced-fit (IF) mechanism,
in which cognate lysine ligand first binds to the riboswitch before
structural rearrangement takes place. The transition state to folding
is found to be enthalpically favored (ΔHfold‡ < 0), yet with a free-energy barrier
that is predominantly entropic (−TΔSfold‡ > 0), which results
in folding (unfolding) rate constants strongly dependent (independent)
of lysine concentration. Analysis of the single-molecule kinetic “trajectories”
reveals this rate constant dependence of kfold on lysine to be predominantly entropic in nature, with the additional
lysine conferring preferential advantage to the folding process by
the presence of ligands correctly oriented with respect to the riboswitch
platform. By way of contrast, van’t Hoff analysis reveals enthalpic
contributions to the overall folding thermodynamics (ΔH0) to be surprisingly constant and robustly
independent of lysine concentration. The results demonstrate the crucial
role of hydrogen bonding between the ligand and riboswitch platform
but with only a relatively modest fraction (45%) of the overall enthalpy
change needed to access the transition state and initiate transcriptional
switching.