The Kinetics of Ligand Binding by an Adenine-Sensing Riboswitch

Abstract

A riboswitch within the 5' untranslated region (UTR) of the Bacillus subtilis pbuE mRNA binds adenine and related analogues in the absence of protein factors; excess adenine added to bacterial growth media triggers activation of a reporter gene that carries this riboswitch. To assess whether the riboswitch reaches thermodynamic equilibrium, or is operated by the kinetics of ligand binding and RNA transcription, we examined the detailed equilibrium and kinetic parameters for the complex formation between the aptamer domain of this riboswitch and the ligands adenine, 2-aminopurine (2AP), and 2,6-diaminopurine (DAP). Using a fluorescence-based assay, we have confirmed that adenine and 2AP have nearly equal binding affinity, with KD values for 2AP ranging from 250 nM to 3 microM at temperatures ranging from 15 to 35 degrees C, while DAP binds with much higher affinity. The association rate constant, however, favors adenine over DAP and 2AP by 3- and 10-fold, respectively, at 25 degrees C. Furthermore, the rate constants for adenine association and dissociation with the aptamer suggest that the pbuE riboswitch could be either kinetically or thermodynamically controlled depending upon the time scale of transcription and external variables such as temperature. We cite data that suggest kinetic control under certain conditions and illustrate with a model calculation how the system can switch between kinetic and equilibrium control. These findings further support the hypothesis that many riboswitches rely on the kinetics of ligand binding and the speed of RNA transcription, rather than simple ligand affinity, to establish the concentration of metabolite needed to trigger riboswitch function.

Structural features of metabolite-sensing riboswitches

“…This approach has proved to be remarkably successful in establishing the overall secondary structure patterns for these RNA species. Furthermore, monitoring these changes over a range of ligand concentrations has allowed for determination of apparent K D values, which for certain riboswitches have also been validated via several biophysical techniques [33,35,36,[39][40][41]. However, the most reliable secondary structures have been obtained when structural probing data have been combined with the comparative sequence analyses of riboswitches from different species.…”

Nucleation of an Allosteric Response via Ligand-induced Loop Folding

“…Consequently, allosteric control operates in a broad range of macromolecules including oligomeric metabolic enzymes (aspartate transcarbamylase)(3) chaperonin machines (GroEL)(4), transcription regulatory proteins (lactose repressor)(5), multi-domain signal transduction proteins (N-WASP) (6), and small molecule-activated riboswitches (Adenine riboswitch) (7). Understanding allostery requires elucidation of the ApoBirA, the unliganded form of BirA, is monomeric, as evidenced by its crystal structure and gel filtration and sedimentation equilibrium studies (14).…”

Le riborégulateur adénine

“…Par exemple, des études in vivo ont montré que ce riborégulateur modulait positivement l'expression d'un gène rapporteur chez B. subtilis en présence d'une concentration suffisante d'adénine [24]. En revanche, des expériences in vitro ont montré que la séquence complète du riborégulateur ne pouvait lier de façon productive l'adénine [38,41]. Ces résultats en apparente contradiction avec le concept général de réversibilité des riborégula-teurs ont été publiés dans deux articles récents [38,39].…”

“…En revanche, des expériences in vitro ont montré que la séquence complète du riborégulateur ne pouvait lier de façon productive l'adénine [38,41]. Ces résultats en apparente contradiction avec le concept général de réversibilité des riborégula-teurs ont été publiés dans deux articles récents [38,39]. Ces études montrent que la vitesse à laquelle la transcription est effectuée joue un rôle prépondérant dans le mécanisme de régulation du riborégulateur.…”

“…La mutation de ces sites de pause engendre une diminution de la formation du complexe ligand-aptamère [39]. En ce qui concerne le riborégulateur adénine, deux sites de pause ont été prédits, ce qui expliquerait la dépendance de la régulation génétique envers la cinétique de liaison du ligand et la vitesse de transcription [38].…”