**3. Identification of RNA thermometers**

#### **3.1.** *In silico* **predictions**

Given that the function of an RNAT is dependent on its structure, the identification of a new RNAT often starts with *in silico* analyses aimed at predicting secondary structure and the Gibbs free energy released by folding of known transcripts at different temperatures. Such predic‐ tions are often generated using freely available web-based programs, such as Mfold and are typically carried out individually for each transcript under investigation [17]. The identifica‐ tion of putative RNATs has been facilitated by the generation of a searchable database that contains the predicted structures within the untranslated regions of bacteria transcripts (RNA-SURIBA) [18]. Additionally, web servers such as RNAtips and RNAthermsw are now availa‐ ble, which calculate the folding energy of a given RNA molecule under varied temperatures [19,20]. Together, these databases and programs can be utilized to predict the presence of a putative RNAT within a given transcript or genome. While *in silico* approaches have proven powerful in the identification of many RNATs, they are limited in that they are only predic‐ tions. Additionally, all currently available *in silico* prediction tools are based on our current understanding of identified RNATs and therefore may not recognize novel types of RNATs with unique structural features. As the number of characterized RNATs continues to grow, so will our ability to accurately predict their presence in sequenced transcripts.

#### **3.2. Identification with experimental approaches**

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‐

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

Given that the function of an RNAT is dependent on its structure, the identification of a new RNAT often starts with *in silico* analyses aimed at predicting secondary structure and the Gibbs free energy released by folding of known transcripts at different temperatures. Such predic‐ tions are often generated using freely available web-based programs, such as Mfold and are typically carried out individually for each transcript under investigation [17]. The identifica‐ tion of putative RNATs has been facilitated by the generation of a searchable database that contains the predicted structures within the untranslated regions of bacteria transcripts (RNA-SURIBA) [18]. Additionally, web servers such as RNAtips and RNAthermsw are now availa‐ ble, which calculate the folding energy of a given RNA molecule under varied temperatures [19,20]. Together, these databases and programs can be utilized to predict the presence of a putative RNAT within a given transcript or genome. While *in silico* approaches have proven powerful in the identification of many RNATs, they are limited in that they are only predic‐

that of the ribosome itself, remains the subject of active investigation.

**3. Identification of RNA thermometers**

**3.1.** *In silico* **predictions**

witches.

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Regardless of the approach used to predict the existence of a functional RNAT, the thermo‐ sensing regulatory activity of each putative element must be validated experimentally. There are several lab-based approaches currently being utilized to demonstrate the functionality of newly identified RNATs. One way in which the thermoresponsive regulatory activity of a putative RNAT is tested is to clone the element being investigated between a constitutive or an arabinose-inducible plasmid promoter and a reporter gene (e.g., *lacZ* or *gfp*) on a plasmid [10,11,21]. By introducing the constructed reporter plasmid into a bacterial strain and meas‐ uring the relative amounts of both the reporter transcript and reporter protein following growth of the strain at different temperatures, the functionality of the putative RNAT under investigation can be accessed. Specifically, if the putative RNAT is functional, production of the reporter protein will increase with the rise of temperature, while the relative levels of reporter transcript will not vary. Such experimental investigations, along with mutagenesis analysis, have been utilized to demonstrate the functionality of several newly identified RNATs [11,22–24] (Figure 2).

**Figure 2.** *In vivo* reporter plasmid-based assay used to experimentally test the thermoresponsive regulatory activity of a putative RNA thermometer. The reporter plasmid is constructed by cloning the inhibitory hairpin of a putative RNA thermometer between a constitutive or arabinose-induced plasmid promoter and a reporter gene. If the RNA ther‐ mometer is functional, it is expected that, following the introduction of the reporter plasmid into a bacterial strain and the growth of that strain at different temperatures, the relative amounts of the reporter transcript will be constant, while the relative levels of the reporter protein will be regulated in response to temperature.

To further validate the functionality of a predicted RNAT, *in vitro* analysis such as structure probing assays can be utilized to directly investigate the impact of varied temperature on the secondary structure of the element under investigation [22,25]. The principle underlying structure probing-based analyses is that specific RNA-digesting enzymes cleave RNA molecules based on the presence of specific secondary structures and/or primary sequences. For example, RNase T1 cleaves immediately 3′ to a single-stranded guanine, while RNase V1 cleaves double-stranded RNA in a sequence-independent manner. Briefly, to perform a structure probing analysis, the putative RNAT under investigation is synthesized by *in vitro* transcription and then radiolabeled at the 5′ end. Next, the labeled RNA molecule is subjected to partial digestion with various RNA-digesting enzymes separately, and the generated fragments visualized by electrophoresis in a denaturing polyacrylamide gel. By completing this analysis at different temperatures it is possible to determine the impact of environmental temperature on the global structure of the RNA molecule (Figure 3).

**Figure 3.** Structure probing assay used to experimentally determine the secondary structure of a putative RNA ther‐ mometer. An *in vitro* transcribed putative RNA thermometer is represented as the hairpin structure in this figure. Fol‐ lowing radioactive end-labeling (indicated by a red star), the molecule is subject to partial digestions with different RNA-digesting enzymes and the resulting products are visualized by electrophoresis. In this figure, enzyme RNase T1 and RNase V1 are presented as examples of RNA-digesting enzymes, which cut, respectively, immediately 3′ to a sin‐ gle-stranded guanine and at double-stranded RNA in a sequence-independent manner. If the putative RNA thermom‐ eter under investigation changes conformation in response to alterations in temperature, an increase of environmental temperature would destabilize the inhibitory hairpin, resulting in a different pattern of radiolabeled fragments follow‐ ing digestion with the RNA degrading enzymes.

Moreover, techniques that study the physical properties of an RNA molecule, such as the nuclear magnetic resonance (NMR) spectroscopy and UV melting analysis, can be utilized to investigate the detailed base-pairing and their changes in response to temperature, thus revealing structural information as well as the molecular basis of thermosensing [7,26]. Together, these experimental analyses provide information about the dynamics of the inhibitory structure of a putative RNAT.

In addition to studies aimed at characterizing temperature-dependent changes in secondary structure, the regulatory activity of putative RNATs can be verified using a toe-printing assay, an *in vitro* analysis designed to directly assess the ability a ribosome to assemble and bind to the SD sequence contained on a given RNA molecule [22,27]. In the case of a functional RNAT, it would be predicted that ribosomal binding occurs at permissive temperatures, when the inhibitory structure is absent, but does not occur at non-permissive temperatures, when the inhibitory structure is present. Briefly, a putative RNAT is synthesized by *in vitro* transcription and incubated at a given temperature with a mixture of ribosome subunits and methionine conjugated tRNAs. If an SD sequence is available, a stable initiation complex will form on the RNA molecule, the presence of which is detected by reverse transcription using a radiolabeled primer that binds the RNA molecule downstream to the ribosome-binding site. The presence of the initiation complex will hinder the progression of the reverse transcriptase and thus result in the formation of a relatively short radiolabeled cDNA product. If the initiation complex cannot be formed, in this case because the SD sequences are occluded by the formation of an inhibitory structure within the putative RNAT, reverse transcription will not be blocked and a relatively long radiolabeled cDNA product will be generated. By completing toe-printing assays at different temperatures, the impact of temperature on the ability of the ribosome to interact with a putative RNAT can be directly determined. In the case of a functional RNAT, it would be expected that a relatively short cDNA product will be formed at permissive temperatures when the transcription initiation complex can form and that a relatively long cDNA product will be formed at non-permissive temperatures when assembling of the translation initiation complex is blocked by the formation of the inhibitory structures of the RNAT (Figure 4).

#### **3.3. RNA structuromics**

To further validate the functionality of a predicted RNAT, *in vitro* analysis such as structure probing assays can be utilized to directly investigate the impact of varied temperature on the secondary structure of the element under investigation [22,25]. The principle underlying structure probing-based analyses is that specific RNA-digesting enzymes cleave RNA molecules based on the presence of specific secondary structures and/or primary sequences. For example, RNase T1 cleaves immediately 3′ to a single-stranded guanine, while RNase V1 cleaves double-stranded RNA in a sequence-independent manner. Briefly, to perform a structure probing analysis, the putative RNAT under investigation is synthesized by *in vitro* transcription and then radiolabeled at the 5′ end. Next, the labeled RNA molecule is subjected to partial digestion with various RNA-digesting enzymes separately, and the generated fragments visualized by electrophoresis in a denaturing polyacrylamide gel. By completing this analysis at different temperatures it is possible to determine the impact of environmental

**Figure 3.** Structure probing assay used to experimentally determine the secondary structure of a putative RNA ther‐ mometer. An *in vitro* transcribed putative RNA thermometer is represented as the hairpin structure in this figure. Fol‐ lowing radioactive end-labeling (indicated by a red star), the molecule is subject to partial digestions with different RNA-digesting enzymes and the resulting products are visualized by electrophoresis. In this figure, enzyme RNase T1 and RNase V1 are presented as examples of RNA-digesting enzymes, which cut, respectively, immediately 3′ to a sin‐ gle-stranded guanine and at double-stranded RNA in a sequence-independent manner. If the putative RNA thermom‐ eter under investigation changes conformation in response to alterations in temperature, an increase of environmental temperature would destabilize the inhibitory hairpin, resulting in a different pattern of radiolabeled fragments follow‐

Moreover, techniques that study the physical properties of an RNA molecule, such as the nuclear magnetic resonance (NMR) spectroscopy and UV melting analysis, can be utilized to investigate the detailed base-pairing and their changes in response to temperature, thus revealing structural information as well as the molecular basis of thermosensing [7,26].

temperature on the global structure of the RNA molecule (Figure 3).

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ing digestion with the RNA degrading enzymes.

Recently, a combination of experimental and next-generation high-throughput techniques have been used to identify the structures of every RNA molecule within a single organism, collectively termed the "RNA structurome" [28]. Structuromic analyses performed at various temperatures have the potential to reveal a massive amount of information that will directly lead to the discovery of potentially expansive numbers of temperature-responsive regulatory RNA elements including RNATs [2]. The structurome of *Saccharomyces cerevisiae* and that of mice nuclear transcriptome were generated using parallel analysis of RNA structure (PARS) and fragmentation sequencing (Frag-seq), respectively [29,30]. The general experimental procedure that reveals the structurome of an organism includes two main steps: 1) structural probing of a certain transcriptome by specific RNA-digesting enzymes or chemicals that differentially cleave or modify RNA molecules based on the presence of specific secondary structures and 2) high-throughput sequencing analysis of the cDNA libraries generated from the digested/modified transcriptome. The structural probing portion of the analysis can be done either *in vitro* by treating the transcriptome harvested from the organism with structureand sequence-specific RNA endonucleases or *in vivo* by cell-penetrating chemicals that modify

**Figure 4.** Toe-printing assay used to experimentally determine differential binding of the ribosome to a putative RNA thermometer at different temperatures. An *in vitro* transcribed putative RNA thermometer is incubated with a mixture of ribosome subunits and methionine-conjugated tRNA under different temperatures and then used as template in a reverse transcription reaction with a radiolabeled primer that binds downstream of the SD sequence. If the ribosome differentially binds the transcript, as would be predicted for that containing a functional RNA thermometer, reverse transcription would be expected to be hindered by the presence of the bound ribosome under permissive temperature (37°C in this example), thus producing a relatively short radiolabeled cDNA product. At non-permissive temperatures (25°C in this example), however, the ribosome would not be bound and reverse transcriptase would be expected to process to the end of the transcript generating a relatively long radiolabeled cDNA product.

or cleave single-stranded RNA bases within the cells [28,31]. This new experimental approach not only provides structural information of RNAs in physiological context but also evades the disadvantages of current *in silico* and *in vitro* analyses. Specifically, *in silico* prediction of RNA secondary structure is dependent on the length of RNA molecule that has been chosen; the longer the sequence, the lower the reliability of the prediction [32]. Additionally, secondary structures characterized by *in vitro* experimental analysis carry the caveat that the structures may be different *in vivo*. It is expected that, by completion of RNA structuromic analyses in a variety of bacterial organisms, the recognized numbers and types of RNATs will grow dramatically, an advancement that is critical to revealing the full impact of RNATs in control‐ ling the physiology and virulence of bacterial species.

## **4. Families of RNA thermometers**

The thermosensing activity of an RNAT is largely dependent on the physical features of its secondary structure, specifically by those features that impact the stability, or the Tm, of the inhibitory hairpin. In addition to the base-stacking interactions and the hydration shell of an RNA helix, other critical features of RNATs include 1) the number and stability of hairpins that are formed within the element; 2) the presence of canonical and non-canonical basepairing within the inhibitory structure; 3) the existence of internal loops, bulges, or mismatches within the formed structure(s); and 4) the extent of base-pairing between sequences composing the SD site and/or start codon with upstream sequences contained on the transcript. Each of these features can directly impact the stability of the inhibitory structure within a given RNAT, which in turn dictates the responsiveness of the element to temperature. Despite sharing a common basic regulatory mechanism, differences in RNATs display different secondary structures and other key features, differences that are now used to classify bacterial RNATs into families. The two currently recognized families of RNATs are ROSE-like RNATs (repres‐ sion of heat shock gene expression) and FourU RNATs. RNATs composing each of these two main families, as well as a few unique RNATs, are discussed below.

#### **4.1. ROSE-like RNA thermometers**

or cleave single-stranded RNA bases within the cells [28,31]. This new experimental approach not only provides structural information of RNAs in physiological context but also evades the disadvantages of current *in silico* and *in vitro* analyses. Specifically, *in silico* prediction of RNA secondary structure is dependent on the length of RNA molecule that has been chosen; the longer the sequence, the lower the reliability of the prediction [32]. Additionally, secondary structures characterized by *in vitro* experimental analysis carry the caveat that the structures may be different *in vivo*. It is expected that, by completion of RNA structuromic analyses in a variety of bacterial organisms, the recognized numbers and types of RNATs will grow dramatically, an advancement that is critical to revealing the full impact of RNATs in control‐

process to the end of the transcript generating a relatively long radiolabeled cDNA product.

**Figure 4.** Toe-printing assay used to experimentally determine differential binding of the ribosome to a putative RNA thermometer at different temperatures. An *in vitro* transcribed putative RNA thermometer is incubated with a mixture of ribosome subunits and methionine-conjugated tRNA under different temperatures and then used as template in a reverse transcription reaction with a radiolabeled primer that binds downstream of the SD sequence. If the ribosome differentially binds the transcript, as would be predicted for that containing a functional RNA thermometer, reverse transcription would be expected to be hindered by the presence of the bound ribosome under permissive temperature (37°C in this example), thus producing a relatively short radiolabeled cDNA product. At non-permissive temperatures (25°C in this example), however, the ribosome would not be bound and reverse transcriptase would be expected to

The thermosensing activity of an RNAT is largely dependent on the physical features of its secondary structure, specifically by those features that impact the stability, or the Tm, of the inhibitory hairpin. In addition to the base-stacking interactions and the hydration shell of an RNA helix, other critical features of RNATs include 1) the number and stability of hairpins that are formed within the element; 2) the presence of canonical and non-canonical basepairing within the inhibitory structure; 3) the existence of internal loops, bulges, or mismatches within the formed structure(s); and 4) the extent of base-pairing between sequences composing

ling the physiology and virulence of bacterial species.

**4. Families of RNA thermometers**

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ROSE-like elements were first identified as conserved *cis*-regulatory elements located in the regions between the promoters and start codons of genes encoding small heat shock proteins (sHsps) in *Bradyrhizobium japonicum*. and within a short time were reported in other *Rhizobi‐ um* species as well as in *Agrobacterium tumefaciens* [21,33–35]. The heat shock response is a highly conserved process among microorganisms, and while their numbers vary between organisms, small heat shock proteins play a critical role in preventing protein denaturation and aggregation under heat stress. Based on the conservation of the biological process as well as the conservation of the primary sequence and secondary structure of the 17 originally identified ROSE-like elements, bioinformatics-based techniques were used to predict ROSElike elements in the 5′ UTRs of sHsp encoding genes from 120 different archaea and bacteria [21,36]. As a result of these studies, 27 additional ROSE-like elements were identified in 18 different α- and γ-proteobacteria species [36]. Likely as a result of the approaches used to identify them, nearly all ROSE-like elements identified to date control the production of factors involved in the heat shock response. However, as additional ROSE-like RNATs are identified and characterized, it is expected that the contribution of these regulatory elements will be expanded beyond the production of heat shock response and into other physiological proc‐ esses. This notion is supported by the recent identification of a ROSE-like RNAT in *Pseudomonas aeruginosa* that controls the production of rhamnolipids, a virulence factor that functions to protect the pathogen against killing by the human immune system [37]. Only with additional studies will the potentially expansive role of ROSE-like thermometers in controlling the physiology and virulence of bacterial species be revealed.

TheROSE-like familyis themost extensivelystudiedfamilyofRNATs,harboringapproximate‐ ly 70% of allRNATs identified to date. AllRNATs within theROSE-like family are housed with 5′ UTR regions that range from 60 nucleotides to more than 100 nucleotides in length and that form 2 to 4 hairpins [36,37]. Within these hairpins, the 5′-proximal hairpin generally acts to stabilize the secondary structure and facilitate the correct folding of the other hairpins, while the 3′-proximal hairpin contains the SD region of the regulated transcript [7]. The defining features of ROSE-like RNATs that contribute to their temperature-responsive regulatory function include 1) the presence of a conserved anti-SD sequence 5′-UYGCU-3′ (Y stands for a pyrimidine) in the 3′-proximal hairpin, and 2) a "bulged" guanine within the SD sequester‐ ing hairpin (Figure 5) [36]. As a feature shared by all ROSE-like elements, it has been pro‐ posed that the "bulged" guanine within the SD sequestering hairpin is essential for the

thermoresponsiveness of the regulatory element, a prediction that is supported by various mutagenesis-basedexperimentalapproachesandbyNMRspectroscopy[7,38,39].Thesestudies havenotonlydemonstratedthatthe"budged"guanineisessentialforfunctionbutalsorevealed that the "bulged" guanine forms hydrogen bonds with the second guanine within the SD sequence of 5′-AGGA-3′. Additionally, towards the 3′ end ofthe SD site, two pyrimidines from the anti-SD strand form a triple-base pair with a uracil from the SD site with hydrogen bonds (Figure 5). The existence of two highly unstable pairs — a G-G pair and a triple-base pair within the inhibitory hairpin of ROSE-like RNATs enables it to respond to the subtle changes of environmental temperature and thus to function as a temperature-sensitive regulatory element [7].

**Figure 5.** Structural features of the ROSE-like family of RNA thermometers, demonstrated by a schematic of the *hspA* RNA thermometer of *B. japonicum*. Within the four hairpins of *hspA* RNA thermometer, only the conserved structural features in the 3′ proximal hairpin are shown in detail, with the varied number of upstream hairpins indicated by the general hairpin structure in parentheses. The red line indicates the location of the SD sequence, while the conserved G-G pairing and triple-base pair are highlighted by the green boxes.

#### **4.2. FourU RNA thermometers**

FourU RNATs, so named due to the presence of four consecutive uracil residues within the SD sequestering inhibitory hairpin, represent the second family of currently identified RNATs. First identified in *Salmonella enterica,* a total of eight FourU RNATs have now been identified and characterized in a variety of bacterial species [10,11,22,40–42]. Unlike ROSE-like RNATs, only two characterized FourU RNATs function to control the production of a heat shockrelated factor [10,22]. Instead, the majority of characterized FourU RNATs (*toxT* from *Vibrio cholera*, *lcrF/virF* from *Yersinia* species, as well as *shuA* from *S. dysenteriae* and its homologous gene *chuA* from some pathogenic *E. coli*) function to regulate the production of virulence factors in response to alterations in environmental temperature [11,40,41].

thermoresponsiveness of the regulatory element, a prediction that is supported by various mutagenesis-basedexperimentalapproachesandbyNMRspectroscopy[7,38,39].Thesestudies havenotonlydemonstratedthatthe"budged"guanineisessentialforfunctionbutalsorevealed that the "bulged" guanine forms hydrogen bonds with the second guanine within the SD sequence of 5′-AGGA-3′. Additionally, towards the 3′ end ofthe SD site, two pyrimidines from the anti-SD strand form a triple-base pair with a uracil from the SD site with hydrogen bonds (Figure 5). The existence of two highly unstable pairs — a G-G pair and a triple-base pair within the inhibitory hairpin of ROSE-like RNATs enables it to respond to the subtle changes of environmental temperature and thus to function as a temperature-sensitive regulatory

**Figure 5.** Structural features of the ROSE-like family of RNA thermometers, demonstrated by a schematic of the *hspA* RNA thermometer of *B. japonicum*. Within the four hairpins of *hspA* RNA thermometer, only the conserved structural features in the 3′ proximal hairpin are shown in detail, with the varied number of upstream hairpins indicated by the general hairpin structure in parentheses. The red line indicates the location of the SD sequence, while the conserved G-

FourU RNATs, so named due to the presence of four consecutive uracil residues within the SD sequestering inhibitory hairpin, represent the second family of currently identified RNATs. First identified in *Salmonella enterica,* a total of eight FourU RNATs have now been identified and characterized in a variety of bacterial species [10,11,22,40–42]. Unlike ROSE-like RNATs,

G pairing and triple-base pair are highlighted by the green boxes.

**4.2. FourU RNA thermometers**

element [7].

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**Figure 6.** Structural features of the FourU family of RNA thermometers, demonstrated by a schematic of the *shuA* RNA thermometer of *S. dysenteriae*. Only the conserved portion of the inhibitory hairpin of *shuA* RNA thermometer is shown in this figure. The red line indicates the location of the SD sequences, while a green box indicates the location of the conserved four consecutive uracil residues.

The structural features of FourU RNATs are largely varied. For example, the length of the 5′ UTRs in which FourU RNATs are housed ranges from as short as 40 nucleotides (*htrA* from *E. coli* and *Salmonella*) to more than 280 nucleotides (*shuA* from *Shigella dysenteriae*) in length [10,11]. Additionally, the number of hairpins varies from a single hairpin with internal loops (*toxT* from *Vibrio cholerae*) to five hairpins, including an inhibitory hairpin with no internal loops (*shuA* from *S. dysenteriae*) [11,40]. Despite these differences, there are also several key features shared within FourU RNATs. The first shared feature is the presence of four consec‐ utive uridine residues that form canonical A-U and/or non-canonical G-U base-pairs with SD sequences on the regulated transcript (Figure 6). Additionally, for RNATs within the FourU family, the SD sequestering hairpin is generated by no less than 5 continuous base-pairs, and often displaying conserved destabilizing features including the presence of relatively few G-C pairs, as well as internal mismatches or loops within the inhibitory structures. Likely due to the innate stability of the inhibitory hairpin within FourU RNATs, features destabilizing the inhibitory structure increase the responsiveness of FourU RNATs to temperature alterations, and disruption of these features result in altered thermosensing abilities. In the studies of each characterized FourU RNAT, mutagenesis analyses that introduce G-C base-pairs into the inhibitory structure result in the expected stabilization and, importantly, loss of thermosensing activity by the regulatory element [11,22,40]. NMR spectroscopy analysis has been utilized to study the dynamics of the inhibitory hairpin within the *agsA* FourU RNAT [15]. Specifically, a point mutation that introduces a C-G base-pair at the previously mismatched position adjacent to the SD region increased the melting temperature of the hairpin by 11°C. Addition‐ ally, two Mg2+ binding sites were found in the *agsA* FourU thermometer hairpin and it was demonstrated that Mg2+ functions to stabilize the inhibitory structure [15]. The degree to which these important features are conserved among members of the FourU RNAT family will be revealed only after additional members are identified and experimentally characterized. Such experimentation will not only define the FourU RNAT family of regulators but will also advance our ability to identify new FourU RNATs.

#### **4.3. Additional types of RNA thermometers**

It is important to note that not all characterized RNATs fit neatly into one of the two main families: ROSE-like and FourU. While all RNATs are thought to share a basic zipper-like thermosensing mechanism, several identified RNATs differ from those composing the main families in critical features, including primary sequence and/or secondary structure, features that impact the regulatory activity of these elements. It is the identification and characterization of the details of the molecular mechanisms underlying each of these additional types of RNATs that will expand our understanding of foundational principles governing RNA-mediated thermosensing.

In some RNATs, base-pairing involving the SD sequence is not complete but instead is disrupted by mismatches or "bulged" nucleotides, a feature also noted for ROSE-like elements. For example, the inhibitory structure within the RNATs that control the production of two putative lipoproteins LigA and LigB in *Leptospira interrogans* have identical nucleic acid sequences that include a mismatch of an adenine and a guanine within the SD sequestering hairpin [43]. Genes *hspX* and *hspY* that encode sHsps in *Pseudomonas putida* are also regulated by RNATs that contain one or two A·G mismatches disrupting the otherwise continued basepairing of the SD region [8]. For these RNATs, further investigation is needed to understand the direct impact of the apparently conserved feature of mismatched or bulged sequences within the inhibitory structure on the regulatory activity of these elements.

Although lacking the presence of four consecutive uracil residues, two RNATs are similar to FourU RNATs in that they display more than 5 continuous base-pairs within the SD region of their inhibitory hairpins: one RNAT controls the production of an sHsp (Hsp17) from *Synechocystis* sp. PCC 6803, while the other controls the production of *Salmonella* GroES, a component of protein chaperon machinery [23,44]. RNAT-mediated regulation of *hsp17* is important for the survival of *Synechocystis* under heat stress, because Hsp17 not only prevents denatured proteins from aggregation but also protects the integrity of cellular membranes [45,46]. The 5′ UTR of *hsp17* has a single hairpin with an internal asymmetric loop [23]. In the SD sequence-binding region, instead of four uracils as seen in the FourU thermometer, the *hsp17* RNAT has a sequence of 5′-UCCU-3′ that forms four canonical pairs with the SD sequence, including two G-C pairs. The remaining base-pairs in the inhibitory hairpin are mainly A-U pairs with two non-canonical G-U pairs. As the most stabled base-pairs within the *hsp17* RNAT, these two G-C base-pairs contribute to the stability and thus the inhibitory function of the hairpin. Other features such as the asymmetric internal loop and low ratio of G-C base-pairs destabilize the inhibitory structure, features that together enable the hairpin to dissociate with the increase of temperature. For the inhibitory hairpin of the *groES* RNAT, it has a mismatch of an adenine and a guanine that destabilizes this structure. While the secondary structure and temperature-responsive regulatory function of the *groES* RNAT has only been experimentally characterized in *Salmonella* and *E. coli*, this RNAT and its regulated factor, a necessary chaperon for proper folding of cellular components, are well conserved in enterobacteria [44].

characterized FourU RNAT, mutagenesis analyses that introduce G-C base-pairs into the inhibitory structure result in the expected stabilization and, importantly, loss of thermosensing activity by the regulatory element [11,22,40]. NMR spectroscopy analysis has been utilized to study the dynamics of the inhibitory hairpin within the *agsA* FourU RNAT [15]. Specifically, a point mutation that introduces a C-G base-pair at the previously mismatched position adjacent to the SD region increased the melting temperature of the hairpin by 11°C. Addition‐ ally, two Mg2+ binding sites were found in the *agsA* FourU thermometer hairpin and it was demonstrated that Mg2+ functions to stabilize the inhibitory structure [15]. The degree to which these important features are conserved among members of the FourU RNAT family will be revealed only after additional members are identified and experimentally characterized. Such experimentation will not only define the FourU RNAT family of regulators but will also

It is important to note that not all characterized RNATs fit neatly into one of the two main families: ROSE-like and FourU. While all RNATs are thought to share a basic zipper-like thermosensing mechanism, several identified RNATs differ from those composing the main families in critical features, including primary sequence and/or secondary structure, features that impact the regulatory activity of these elements. It is the identification and characterization of the details of the molecular mechanisms underlying each of these additional types of RNATs that will expand our understanding of foundational principles governing RNA-mediated

In some RNATs, base-pairing involving the SD sequence is not complete but instead is disrupted by mismatches or "bulged" nucleotides, a feature also noted for ROSE-like elements. For example, the inhibitory structure within the RNATs that control the production of two putative lipoproteins LigA and LigB in *Leptospira interrogans* have identical nucleic acid sequences that include a mismatch of an adenine and a guanine within the SD sequestering hairpin [43]. Genes *hspX* and *hspY* that encode sHsps in *Pseudomonas putida* are also regulated by RNATs that contain one or two A·G mismatches disrupting the otherwise continued basepairing of the SD region [8]. For these RNATs, further investigation is needed to understand the direct impact of the apparently conserved feature of mismatched or bulged sequences

Although lacking the presence of four consecutive uracil residues, two RNATs are similar to FourU RNATs in that they display more than 5 continuous base-pairs within the SD region of their inhibitory hairpins: one RNAT controls the production of an sHsp (Hsp17) from *Synechocystis* sp. PCC 6803, while the other controls the production of *Salmonella* GroES, a component of protein chaperon machinery [23,44]. RNAT-mediated regulation of *hsp17* is important for the survival of *Synechocystis* under heat stress, because Hsp17 not only prevents denatured proteins from aggregation but also protects the integrity of cellular membranes [45,46]. The 5′ UTR of *hsp17* has a single hairpin with an internal asymmetric loop [23]. In the SD sequence-binding region, instead of four uracils as seen in the FourU thermometer, the *hsp17* RNAT has a sequence of 5′-UCCU-3′ that forms four canonical pairs with the SD

within the inhibitory structure on the regulatory activity of these elements.

advance our ability to identify new FourU RNATs.

**4.3. Additional types of RNA thermometers**

168 Nucleic Acids - From Basic Aspects to Laboratory Tools

thermosensing.

For some RNATs, the function and stability of the inhibitory hairpin are impacted by basepairing with sequences other than those within the SD region. For example, in the 5′ UTR of *prfA* from *Listeria monocytogenes*, a major portion of the SD region and the start codon are confined within internal loops and thus are partially single-stranded [47]. It has been demon‐ strated, however, that the hairpin within the *prfA* 5′ UTR containing the SD region and start codon does function as an RNA thermometer, an activity that is dependent on base-pairs that are located upstream of the SD site, which function to stabilize the unusually long hairpin. Another example of sequences other than those within the SD region that directly impact the regulatory function of an RNAT is the repeated nucleotide sequence of 5′-UAUACUUA-3′ in the RNAT of *cssA* from *Neisseria meningitides* [24]. These 8-nucleotide sequences are located upstream of the SD region and enable the RNAT to sense mild changes of environmental temperature, which is important for the survival of *N. meningitides.* As an opportunistic pathogen that colonizes only humans, it is important that *N. meningitidis* can sense and respond to a mild increase of temperature, as would be encountered during a fever response.

A unique example among currently identified RNATs is the one that controls the expression of *rpoH* in *E. coli* [9]. Binding of the ribosome to the SD region within the *ropH* transcript is facilitated by a sequence (named downstream box) located between the SD site and the start codon [48]. The *rpoH* RNAT inhibits translation via embedding this downstream box in the junction region of three stem loops instead of forming base-pairs within a single inhibitory hairpin as is the usual conformation in RNATs [9]. As the environmental temperature increases, two stems that paired with the downstream box melt at the junction position exposing the downstream box as a single strand, a conformation that facilitates ribosome binding to the transcript.

Lastly, there are currently three characterized RNATs that are located within intergenic regions of a polycistronic transcripts: *ibpB* from *E. coli*, *lcrF* from *Yersinia* species, and *hspY* from *P. putida* [8,41,49]. Their location within polycistronic transcripts differentiates these three RNATs from all others found in the 5′ UTR of monocistronic or polycistronic transcripts.

Although they display key features that differ from those possessed by RNATs in the ROSElike or FourU families, many of the unique RNATs highlighted above are conserved between several bacterial species. There is little doubt that as additional bacterial RNATs are identified and characterized, commonalities will emerge and additional families will be recognized.
