Site-Pecific Fluorescnce Dynamics in an RNA ‘THERMOMETER’ Reveals the Mechanism of Temperature-Sensitive Translation

Abstract

The ROSE (Repression Of heat Shock gene Expression) element of mRNA present in the 5’-UTR of small heat-shock genes in many Gram-negative bacteria is known to function as a ‘RNA thermometer’ by controlling protein translation in a temperature range of 30 - 42° C where the translation is blocked till 30°C and allowed at 42°C and beyond, perhaps due to an unfolding transition of the ROSE hair-pin motif.In this work, we have used site-specific fluorescence labeling and pico-second time-domain fluorescence spectroscopy to unravel the mechanism. The ‘ROSE RNA’ was site-specially labeled with 2-aminopurine (2-AP), a fluorescent analog of adenine. Observables such as fluorescence lifetime, fluorescence anisotropy decay kinetics and dynamic fluorescence quenching revealed properties such as the level of base stacking, rotational motion of the bases, segmental dynamics of the backbone and the level of exposure of base to solvent. As expected, all read-outs of 2-AP residue that were studied showed remarkable position-dependence/sensitivity in the RNA sequence at 25°C. The striking result was the persistence of the same position-dependence of the parameters even at 45°C albeit at a measurably reduced levels. However the same position-dependence was nearly ‘wiped out’ in the presence of urea where all intra-molecular interactions in RNA are undone. These observations have prompted us to revise the existing model of ROSE RNA action: we now suggest that unlike proposed earlier, the thermometer action of ROSE emanates not from its unfolding structural transition between 25 and 45°C, but rather from its propensity to enhance structural dynamics without “melting” the structure. We hypothesize that either the enhanced dynamics of the structure it self or its full melting due to an extrinsic factor (perhaps a protein interaction) might be the basis of its thermometer action.