**2. Molecular mechanism underlying the regulatory function of RNA thermometers**

The molecular mechanism underlying the regulatory activity of RNATs is exquisitely simple, mediated entirely by temperature-induced structural changes within a target mRNA molecule. The currently proposed model of the molecular mechanism underlying RNAT function is that of a zipper [5]. More specifically, at relatively low "non-permissive" temperatures, an inhibi‐ tory structure is formed within the RNAT, at least in part, by binding of Shine-Dalgarno (SD) sequences with upstream sequences within the regulated transcript. Once formed, the inhibitory structure functions to block translation initiation by physically preventing binding of the ribosome to the regulated transcript. With an increase of temperature to that within a permissive range, the base-pairs that stabilize the inhibitory structure within the RNAT gradually dissociate, the ribosome-binding site becomes increasingly exposed and translation proceeds (Figure 1) [7,12].

**Figure 1.** A schematic of the molecular mechanism underlying the regulatory function of RNA thermometers. In the figure, the hairpin structure indicates the inhibitory hairpin of an RNA thermometer, with the red line representing the region of Shine-Dalgarno (SD) sequence or ribosome-binding site. At relatively low, or non-permissive, temperatures, the formation of the inhibitory hairpin inhibits translation from the transcript by preventing binding of ribosome to the SD sequences. As the environmental temperature increases to the permissive range, the inhibitory structure dissoci‐ ates, giving the ribosome access to the SD sequence and thus permitting translation.

Though responsive to temperature, RNAT-mediated regulation is not an all-or-nothing regulation but rather the shifting of an equilibrium towards an open or closed configuration depending on temperature [5]. Furthermore, mutagenesis-based experimentation has clearly demonstrated that it is the altered stability of the inhibitory structure rather than the primary sequence that plays the most critical role in the regulatory function of RNATs [13].

Several features differentiate RNATs from metabolites-binding riboswitches, a superficially related class of ribo-regulators. Firstly, as demonstrated by UV and NMR spectroscopy assays, the temperature-induced destabilization of the inhibitory structure within a given RNAT is a gradual and reversible process [7,14]. As a result of these fundamental features, RNATs mediate a graded response to temperature as opposed to an "on/off" type of regulation that is often associated with riboswitch-mediated regulation. Secondly, unlike riboswitches, structural changes within the RNAT are not mediated by an interaction with a small molecule or other cellular component, a foundational feature confirmed by *in vitro* structural analyses [7]. While the regulatory activity of RNATs is not responsive to the presence or absence of a ligand, Mg2+ has been found to facilitate the regulatory function of some RNATs [15]. Specif‐ ically, Mg2+ has been shown to affect the stability and thus the temperature responsiveness of the inhibitory structure of some RNATs, a feature that is fundamentally different than small molecule-induced switching between mutually exclusive structures as is seen with ribos‐ witches.

While many advances have been made in recent years, several questions remain regarding the details of the molecular mechanism(s) underlying the activity of RNATs. For example, several studies investigating the regulatory mechanism of RNATs focus exclusively on the hairpin containing the SD sequence. As a consequence, the impact of additional structural features within an RNAT, particularly that of commonly observed upstream hairpins, remains largely unknown. Additionally, a recent study revealed that, for at least a subset of RNATs, the ribosome can bind to the SD sequence of the regulated transcript even at non-permissive temperatures when the inhibitory structure would be present [16]. Finally, while the current model of regulation invokes nothing more than temperature in mediating the structural changes that underlie the regulatory activity of RNATs, the role of additional factors, including that of the ribosome itself, remains the subject of active investigation.
