Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit

Abstract

In bacteria, the binding between the riboswitch aptamer domain and ligand is regulated by environmental cues, such as low Mg in macrophages during pathogenesis to ensure spatiotemporal expression of virulence genes. Binding was investigated between the flavin mononucleotide (FMN) riboswitch aptamer and its anionic ligand in the presence of molecular crowding agent without Mg ion, which mimics pathogenic conditions. Structural, kinetic, and thermodynamic analyses under the crowding revealed more dynamic conformational rearrangements of the FMN riboswitch aptamer compared to dilute Mg -containing solution. It is hypothesized that under crowding conditions FMN binds through an induced fit mechanism in contrast to the conformational selection mechanism previously demonstrated in dilute Mg solution. Since these two mechanisms involve different conformational intermediates and rate constants, these findings have practical significance in areas such as drug design and RNA engineering.

Transcriptional Riboswitches Integrate Timescales for Bacterial Gene Expression Control

“…This dependence of the folding pathway on the relative timescales of ligand binding and conformational dynamics of the aptamer can be identified as a kinetic coupling mechanism occurring early in the decision tree of gene regulation (Figure 1B). Similar kinetic control mechanisms of ligand recognition by the aptamer have been observed to be operational in multiple other riboswitches (Manz et al, 2017;Rode et al, 2018;McCluskey et al, 2019;Sung and Nesbitt, 2019).…”

“…The transition between the IF and CS models is governed, on one hand, by the ligand concentration and, on the other hand, by temperature and cofactors affecting RNA folding such as the cationic micro-environment. Undoubtedly, evolutionary pressures shape the sequence composition of the riboswitch to finetune this balance to the cell's needs (Suddala and Walter, 2014;Suddala et al, 2015;Rode et al, 2018). Ligand recognition mechanisms like the CS and IF models have provided the basis for the kinetic selection of transcriptional riboswitches (Suddala and Walter, 2014).…”

Molecular Fitness Landscapes from High-Coverage Sequence Profiling

“…Crowded and confined conditions can modify the structure and function of nucleic acids and proteins (96)(97)(98)(99)(100). High levels of molecular crowding have been shown to stabilize mutations in ribozymes (101), change the binding mechanism of a ligand to a riboswitch (102), and create a chaperoning effect to assist in aptamer folding (99). Ribozymes can also modify their environment (e.g., through cooperation (103)), presenting an attractive future target for mapping more complex fitness landscapes.…”

Divalent Cation Dependence Enhances Dopamine Aptamer Biosensing

“…In addition to structural stabilization of the free state of single-stranded RNA, two competing mechanisms of target recognition have been reported in the presence vs absence of Mg 2+ . 66 Data from fluorescence titration, nuclease digestion, and isothermal titration calorimetry have suggested that without Mg 2+ , a flavin mononucleotide riboswitch interacts with its anionic target via an induced fit mechanism, where target recognition triggers conformational change. 66 Alternately, in the presence of Mg 2+ , this riboswitch undergoes a conformational selection mechanism, where RNA structural rearrangement precedes target binding.…”

“…66 Data from fluorescence titration, nuclease digestion, and isothermal titration calorimetry have suggested that without Mg 2+ , a flavin mononucleotide riboswitch interacts with its anionic target via an induced fit mechanism, where target recognition triggers conformational change. 66 Alternately, in the presence of Mg 2+ , this riboswitch undergoes a conformational selection mechanism, where RNA structural rearrangement precedes target binding. These two mechanisms occur via different conformational intermediates and with different binding rate constants.…”