Site-specific fluorescence dynamics in an RNA 'thermometer' reveals the role of ribosome binding in its temperature-sensitive switch function

摘要

RNA thermometers control the translation of several heat shock and virulence genes by their temperature-sensitive structural transitions. Changes in the structure and dynamics of MiniROSE RNA, which regulates translation in the temperature range of 20-45 degrees C, were studied by site specifically replacing seven adenine residues with the fluorescent analog, 2-aminopurine (2-AP), one at a time. Dynamic fluorescence observables of 2-AP-labeled RNAs were compared in their free versus ribosome-bound states for the first time. Noticeably, position dependence of fluorescence observables, which was prominent at 20 degrees C, was persistent even at 45 degrees C, suggesting the persistence of structural integrity up to 45 degrees C. Interestingly, position-dependent dispersion of fluorescence lifetime and quenching constant at 45 degrees C was ablated in ribosome-bound state, when compared to those at 20 degrees C, underscoring loss of structural integrity at 45 degrees C, in ribosome-bound RNA. Significant increase in the value of mean lifetime for 2-AP corresponding to Shine-Dalgarno sequences, when the temperature was raised from 20 to 45 degrees C, to values seen in the presence of urea at 45 degrees C was a strong indicator of melting of the 3D structure of MiniROSE RNA at 45 degrees C, only when it was ribosome bound. Taken all together, we propose a model where we invoke that ribosome binding of the RNA thermometer critically regulates temperature sensing functions in MiniROSE RNA.