The kinetics of ligand binding by an adenine-sensing riboswitch.
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.
Wickiser
J Kenneth
JK
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8103, USA.
Cheah
Ming T
MT
Breaker
Ronald R
RR
Crothers
Donald M
DM
D015075:2-Aminopurine / Q000031:analogs & derivatives / Q000737:chemistry; D020121:5' Untranslated Regions; D000225:Adenine / Q000737:chemistry*; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D006358:Hot Temperature; D007700:Kinetics; D008024:Ligands; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D001426:Bacterial Proteins; D008024:Ligands; D012333:RNA, Messenger; D015075:2-Aminopurine; C007300:2,6-diaminopurine; D012313:RNA; D000225:Adenine