AnRNA thermometer (orRNA thermosensor) is atemperature-sensitivenon-coding RNA molecule which regulatesgene expression.[1] Its unique characteristic it is that it does not need proteins or metabolites to function, but only reacts to temperature changes.[2] RNA thermometers often regulate genes required during either aheat shock orcold shock response, but have been implicated in other regulatory roles such as inpathogenicity andstarvation.[1]
In general, RNA thermometers operate by changing theirsecondary structure andtertiary structure[3] in response to temperature fluctuations. This structural transition can then expose or occlude important regions of RNA such as aribosome binding site, which then affects thetranslation rate of a nearby protein-codinggene.
RNA thermometers, along withriboswitches, are used as examples in support of theRNA world hypothesis. This theory proposes that RNA was once the solenucleic acid present in cells, and was replaced by the currentDNA → RNA → protein system.[4]
Examples of RNA thermometers includeFourU,[5] theHsp90cis-regulatory element,[6] theROSE element,[7] theLig RNA thermometer,[8] and theHsp17 thermometer.[9]
The first temperature-sensitive RNA element was reported in 1989.[10] Prior to this research, mutations upstream from thetranscription start site in alambda (λ) phage cIIImRNA were found to affect the level of translation of the cIII protein.[11] This protein is involved in selection of either alytic orlysogenic life cycle in λ phage, with high concentrations of cIII promoting lysogeny.[11] Further study of this upstream RNA region identified two alternativesecondary structures; experimental study found the structures to be interchangeable, and dependent on bothmagnesium ion concentration and temperature.[10][12] This RNA thermometer is now thought to encourage entry to a lytic cycle under heat stress in order for thebacteriophage to rapidly replicate and escape the host cell.[1]
The term "RNA thermometer" was not coined until 1999,[13] when it was applied to therpoH RNA element identified inEscherichia coli.[14] More recently,bioinformatics searches have been employed to uncover several novel candidate RNA thermometers.[15] Traditional sequence-based searches are inefficient, however, as the secondary structure of the element is much moreconserved than thenucleic acid sequence.[15]
Biological reactions and organism are sensitive to temperature for cell function. RNA thermometers are an efficient way to respond to temperature because as they allow cells to monitor and sense changes to maintain the cell alive and stable. DNA, RNA, or protein-induced mechanisms avoid small changes because by sensing any external changes[16]
Bacteria use RNA thermometers to enter and survive in their hosts by mounting themselves to their host and causing fluctuations in their temperature. The bacteria can respond quickly against heat-shock and cold-shock conditions since RNA thermometers control gene expression at a translational level.[16]
The first RNA thermometer discovered in chloroplast ofChlamydomonas reinhardtii, found in the 5’-UTR of the psaA mRNA. Its function was different especially because it was considered absent, it has a hairpin-type secondary structure that protects the Shine–Dalgarno sequence when temperature is low, but once a change occurs in temperature, it melts and activates protein production.[2]C. reinhardtii’s RNA thermometer research is the entryway to observing the chloroplast of photosynthetic organisms for gene regulation and how it can be used for agriculture at some point in the future since it helps plants get accustomed to external temperature.[2]
Most known RNA thermometers are located in the5′ untranslated region (UTR) of messenger RNA encodingheat shock proteins—though it has been suggested this fact may be due, in part, tosampling bias and inherent difficulties of detecting short, unconserved RNA sequences ingenomic data.[17][18]
Though predominantly found inprokaryotes, a potential RNA thermometer has been found inmammals includinghumans.[19] The candidate thermosensor heat shock RNA-1 (HSR1) activatesheat-shock transcription factor 1 (HSF1) and induces protective proteins when cell temperature exceeds 37 °C (body temperature), thus preventing the cells from overheating.[19]
The first RNA thermometer discovered in chloroplast of Chlamydomonas reinhardtii, found in the 5’-UTR of the psaA mRNA. Its function was different especially because it was considered absent, it has a hairpin-type secondary structure that protects the Shine–Dalgarno sequence when temperature is low, but once a change occurs in temperature, it melts and activates protein production.[2] C. reinhardtii’s RNA thermometer research is the entryway to observing the chloroplast of photosynthetic organisms for gene regulation and how it can be used for agriculture at some point in the future since it helps plants get accustomed to external temperature.[2]
ROSE elements, are a bacterial RNA thermometer class that regulates the activation of genes that have small heat shock proteins. It melts at a moderate level parallel to the increase of the temperature surrounding its environment. Once it fully melts at a high temperature of ~42 °C, it proceeds to release of Shine–Dalgarno and the AUG start codon. RNA Thermometers can also be found in some plant symbiotes or pathogens, symbiotes and pathogens use the RNA thermometers to regulate the plant's gene expression.[3] A well studied symbiotic bacteria is the Rhizobiaceae family. In majority of the rhizobial species, ROSE elements (cis-acting) were visible controlling heat-shock genes.[3]
RNA thermometers are structurally simple and can be made from short RNA sequences; the smallest is just 44nucleotides and is found in the mRNA of a heat-shock protein, hsp17, inSynechocystis speciesPCC 6803.[9] Generally these RNA elements range in length from 60 to 110 nucleotides[21] and they typically contain ahairpin with a small number of mismatched base pairs which reduce the stability of the structure, thereby allowing easier unfolding in response to a temperature increase.[17]
Detailed structural analysis of the ROSE RNA thermometer revealed that the mismatched bases are actually engaged in nonstandard basepairing that preserves the helical structure of the RNA (see figure). The unusual basepairs consist of G-G, U-U, and UC-U pairs. Since these noncanonical base pairs are relatively unstable, increased temperature causes local melting of the RNA structure in this region, exposing the Shine-Dalgarno sequence.[20]
Some RNA thermometers are significantly more complex than a single hairpin, as in the case of a region found inCspA mRNA which is thought to contain apseudoknot, as well as multiple hairpins.[22][23]
Synthetic RNA thermometers have been designed with just a simple single-hairpin structure.[24] However, thesecondary structure of such short RNA thermometers can be sensitive to mutation, as a singlebase change can render the hairpin inactivein vivo.[25]
RNA thermometers are found in the5′ UTR of messenger RNA, upstream of a protein-coding gene.[1] Here they are able to occlude the ribosome binding site (RBS) and prevent translation of the mRNA into protein.[17] As temperature increases, the hairpin structure can 'melt' and expose the RBS orShine-Dalgarno sequence to permit binding of the small ribosomal subunit (30S), which then assembles other translation machinery.[1] Thestart codon, typically found 8 nucleotides downstream of the Shine-Dalgarno sequence,[17] signals the beginning of a protein-coding gene which is then translated to apeptide product by theribosome. In addition to thiscis-acting mechanism, a lone example of atrans-acting RNA thermometer has been found inRpoS mRNA where it is thought to be involved in the starvation response.[1]
A specific example of an RNA thermometer motif is the FourU thermometer found inSalmonella enterica.[5] When exposed to temperatures above 45 °C, thestem-loop thatbase-pairs opposite the Shine-Dalgarno sequence becomes unpaired and allows the mRNA to enter the ribosome for translation to occur.[25]Mg2+ ion concentration has also been shown to affect the stability of FourU.[26] The most well-studied RNA thermometer is found in therpoH gene inEscherichia coli.[27] This thermosensor upregulates heat shock proteins under high temperatures through σ32, a specialised heat-shocksigma factor.[13]
Though typically associated with heat-induced protein expression, RNA thermometers can also regulate cold-shock proteins.[22] For example, the expression of two 7kDa proteins are regulated by an RNA thermometer in thethermophilic bacteriumThermus thermophilus[28] and a similar mechanism has been identified inEnterobacteriales.[23]
RNA thermometers sensitive to temperatures of 37 °C can be used bypathogens to activate infection-specific genes.[17] For example, theupregulation ofprfA, encoding a key transcriptional regulator ofvirulence genes inListeria monocytogenes, was demonstrated by fusing the5′ DNA ofprfA to thegreen fluorescent protein gene; the gene fusion was then transcribed from the T7 promoter inE. coli, and fluorescence was observed at 37 °C but not at 30 °C.[29]
The RNA world hypothesis states that RNA was once both the carrier of hereditary information andenzymatically active, with different sequences acting as biocatalysts, regulators and sensors.[30] The hypothesis then proposes that modernDNA, RNA and protein-based life evolved andselection replaced the majority of RNA's roles with otherbiomolecules.[4]
RNA thermometers and riboswitches are thought to beevolutionarily ancient due to their wide-scale distribution in distantly-related organisms.[31] It has been proposed that, in the RNA world, RNA thermosensors would have been responsible for temperature-dependent regulation of other RNA molecules.[4][32] RNA thermometers in modern organisms may bemolecular fossils which could hint at a previously more widespread importance in an RNA world.[4]