Wickiser J Kenneth, Cheah Ming T, Breaker Ronald R, Crothers Donald M
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8103, USA.
Biochemistry. 2005 Oct 11;44(40):13404-14. doi: 10.1021/bi051008u.
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.
枯草芽孢杆菌pbuE mRNA的5'非翻译区（UTR）内的核糖开关在没有蛋白质因子的情况下结合腺嘌呤及相关类似物；向细菌生长培养基中添加过量腺嘌呤会触发携带此核糖开关的报告基因的激活。为了评估核糖开关是否达到热力学平衡，或者是由配体结合和RNA转录的动力学所操控，我们研究了该核糖开关的适体结构域与配体腺嘌呤、2-氨基嘌呤（2AP）和2,6-二氨基嘌呤（DAP）之间形成复合物的详细平衡和动力学参数。使用基于荧光的检测方法，我们证实腺嘌呤和2AP具有几乎相等的结合亲和力，在15至35摄氏度的温度范围内，2AP的KD值在250 nM至3 microM之间，而DAP的结合亲和力要高得多。然而，在25摄氏度时，缔合速率常数对腺嘌呤的偏好分别比对DAP和2AP高3倍和10倍。此外，腺嘌呤与适体缔合和解离的速率常数表明，根据转录的时间尺度和诸如温度等外部变量，pbuE核糖开关可能受动力学或热力学控制。我们引用了表明在某些条件下动力学控制的数据，并用模型计算说明了该系统如何在动力学和平衡控制之间切换。这些发现进一步支持了这样的假设，即许多核糖开关依靠配体结合的动力学和RNA转录的速度，而非简单的配体亲和力，来确定触发核糖开关功能所需的代谢物浓度。
