Abstract
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°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°C, was persistent even at 45ºC, suggesting the persistence of structural integrity up to 45ºC. Interestingly, position-dependent dispersion of fluorescence lifetime and quenching constant at 45°C was ablated in ribosome-bound state, when compared to those at 20°C, underscoring loss of structural integrity at 45°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°C, to values seen in the presence of urea at 45°C was a strong indicator of melting of the 3D structure of MiniROSE RNA at 45°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.
Highlights
The information content of an RNA primarily resides in its sequence, the conformation and dynamics of subregions of mRNA are emerging as critical regulators of expression of these messages encoded in the sequence [1,2,3,4,5,6,7]
MiniROSE RNA is the functional domain of an RNA thermometer sequence (Figure 1)
The conclusions drawn were centered mostly on the structural transitions that lead to the opening-up of biologically relevant sites on the MiniROSE RNA, namely the SD sequence and the AUG start codon (Figure 1)
Summary
The information content of an RNA primarily resides in its sequence, the conformation and dynamics of subregions of mRNA are emerging as critical regulators of expression of these messages encoded in the sequence [1,2,3,4,5,6,7]. The seven RNA constructs were used to measure the above-mentioned dynamic fluorescence parameters and explore the temperature-dependent changes in the structure and dynamics vis-a-vis wild-type MiniROSE RNA thermometer in its free versus ribosome-bound states.
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