Genome-wide bioinformatic prediction and experimental evaluation of potential RNA thermometers

Only recently, the fundamental role of regulatory RNAs in prokaryotes and eukaryotes has been appreciated. We developed a pipeline from bioinformatic prediction to experimental validation of new RNA thermometers. Known RNA thermometers are located in the 5′-untranslated region of certain heat shock or virulence genes and control translation by temperature-dependent base pairing of the ribosome binding site. We established the searchable database RNA-SURIBA (Structures of Untranslated Regions In BActeria). A structure-based search pattern reliably recognizes known RNA thermometers and predicts related structures upstream of annotated genes in complete genome sequences. The known ROSE₁ (Repression Of heat Shock gene Expression) thermometer and several other functional ROSE-like elements were correctly predicted. For further investigation, we chose a new candidate upstream of the phage shock gene D (pspD) in the pspABCDE operon of E. coli. We established a new reporter gene system that measures translational control at heat shock temperatures and we demonstrated that the upstream region of pspD does not confer temperature control to the phage shock gene. However, translational efficiency was modulated by a point mutation stabilizing the predicted hairpin. Testing other candidates by this structure prediction and validation process will lead to new insights into the requirements for biologically active RNA thermometers. The database is available on . Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Potential thermosensitive riboswitches in the genome of Salmonella

Currently, a number of structurally and functionally different thermosensitive elements, such as structurally and functionally different RNA thermometers, for controlling a variety of biological processes in bacteria, including virulence are known. These well-known RNA thermometers are structures, whether matched or mismatched, which are represented by either a single stretched hairpin structure or a few hairpins. Based on computer and thermodynamic analyses of 25 isolates of Salmonella enterica with complete genome, we have developed an algorithm and criteria to search for potential RNA thermometers, which will enable us to undertake a future search for potential riboswitches in the genomes of other socially significant pathogens. In addition to the well-known 4U RNA thermometer, another four hairpin-loop structures have been identified in S. enterica as new potential RNA thermometers and two of them are localized in 5′-UTR of virulence regulators gltB and yaeQ. They are highly conserved noncanonical structures and correspond to the necessary and sufficient conditions for forming RNA thermometers, since they are found in each of the 25 S. enterica genome isolates. We analyzed the thermosensitive motif in the pXO1 plasmid of Bacillus anthracis—an anthrax-causative pathogen—and visualized matched hairpins that form a cruciform structure in pUC8 supercoiled plasmid by atomic force microscopy.     

Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli

RNA thermometers are thermosensors that regulate gene expression by temperature-induced changes in RNA conformation. Naturally occurring RNA thermometers exhibit complex secondary structures which are believed to undergo a series of gradual structural changes in response to temperature shifts. Here, we report the de novo design of considerably simpler RNA thermometers that provide useful RNA-only tools to regulate bacterial gene expression by a shift in the growth temperature. We show that a single small stem-loop structure containing the ribosome binding site is sufficient to construct synthetic RNA thermometers that work efficiently at physiological temperatures. Our data suggest that the thermometers function by a simple melting mechanism and thus provide minimum size on/off switches to experimentally induce or repress gene expression by temperature.     

Search for the new potential RNA thermometers in the genome of Salmonella enterica

Microbiology - Currently, a number of structurally and functionally different temperature-sensitive elements such as RNA thermometers which control a variety of biological processes in bacteria,...     

Molecular basis for temperature sensing by an RNA thermometer

Regulatory RNA elements, like riboswitches, respond to intracellular signals by three-dimensional (3D) conformational changes. RNA thermometers employ a similar strategy to sense temperature changes in the cell and regulate the translational machinery. We present here the first 3D NMR structure of the functional domain of a highly conserved bacterial RNA thermometer containing the ribosome binding site that remains occluded at normal temperatures (30 degrees C). We identified a region adjacent to the Shine-Dalgarno sequence that has a network of weak hydrogen bonds within the RNA helix. With the onset of heat shock at 42 degrees C, destabilisation of the RNA structure initiates at this region and favours the release of the ribosome binding site and of the start codon. Deletion of a highly conserved G residue leads to the formation of a stable regular RNA helix that loses thermosensing ability. Our results indicate that RNA thermometers are able to sense temperature changes without the aid of accessory factors.     

Bacterial RNA thermometers: molecular zippers and switches

Bacteria use complex strategies to coordinate temperature-dependent gene expression. Many genes encoding heat shock proteins and virulence factors are regulated by temperature-sensing RNA sequences, known as RNA thermometers (RNATs), in their mRNAs. For these genes, the 5' untranslated region of the mRNA folds into a structure that blocks ribosome access at low temperatures. Increasing the temperature gradually shifts the equilibrium between the closed and open conformations towards the open structure in a zipper-like manner, thereby increasing the efficiency of translation initiation. Here, we review the known molecular principles of RNAT action and the hierarchical RNAT cascade in Escherichia coli. We also discuss RNA-based thermosensors located upstream of cold shock and other genes, translation of which preferentially occurs at low temperatures and which thus operate through a different, more switch-like mechanism. Finally, we consider the potential biotechnological applications of natural and synthetic RNATs.     

Modulation of the stability of the Salmonella fourU-type RNA thermometer

RNA thermometers are translational control elements that regulate the expression of bacterial heat shock and virulence genes. They fold into complex secondary structures that block translation at low temperatures. A temperature increase releases the ribosome binding site and thus permits translation initiation. In fourU-type RNA thermometers, the AGGA sequence of the SD region is paired with four consecutive uridines. We investigated the melting points of the wild-type and mutant sequences. It was decreased by 5°C when a stabilizing GC basepair was exchanged by an AU pair or increased by 11°C when an internal AG mismatch was converted to a GC pair, respectively. Stabilized or destabilized RNA structures are directly correlated with decreased or increased in vivo gene expression, respectively. Mg(2+) also affected the melting point of the fourU thermometer. Variations of the Mg(2+) concentration in the physiological range between 1 and 2 mM translated into a 2.8°C shift of the melting point. Thus, Mg(2+) binding to the hairpin RNA is regulatory relevant. Applying three different NMR techniques, two Mg(2+) binding sites were found in the hairpin structure. One of these binding sites could be identified as outer sphere binding site that is located within the fourU motif. Binding of the two Mg(2+) ions exhibits a positive cooperativity with a Hill coefficient of 1.47. Free energy values ΔG for Mg(2+) binding determined by NMR are in agreement with data determined from CD measurements.     

RNAthermsw: Direct Temperature Simulations for Predicting the Location of RNA Thermometers

The mechanism of RNA thermometers is a subject of growing interest. Also known as RNA thermosensors, these temperature-sensitive segments of the mRNA regulate gene expression by changing their secondary structure in response to temperature fluctuations. The detection of RNA thermometers in various genes of interest is valuable as it could lead to the discovery of new thermometers participating in fundamental processes such as preferential translation during heat-shock. RNAthermsw is a user-friendly webserver for predicting the location of RNA thermometers using direct temperature simulations. It operates by analyzing dotted figures generated as a result of a moving window that performs successive energy minimization folding predictions. Inputs include the RNA sequence, window size, and desired temperature change. RNAthermsw can be freely accessed at http://www.cs.bgu.ac.il/~rnathemsw/RNAthemsw/ (with the slash sign at the end). The website contains a help page with explanations regarding the exact usage.     

Is Thermosensing Property of RNA Thermometers Unique?

A large number of studies have been dedicated to identify the structural and sequence based features of RNA thermometers, mRNAs that regulate their translation initiation rate with temperature. It has been shown that the melting of the ribosome-binding site (RBS) plays a prominent role in this thermosensing process. However, little is known as to how widespread this melting phenomenon is as earlier studies on the subject have worked with a small sample of known RNA thermometers. We have developed a novel method of studying the melting of RNAs with temperature by computationally sampling the distribution of the RNA structures at various temperatures using the RNA folding software Vienna. In this study, we compared the thermosensing property of 100 randomly selected mRNAs and three well known thermometers - rpoH, ibpA and agsA sequences from E. coli. We also compared the rpoH sequences from 81 mesophilic proteobacteria. Although both rpoH and ibpA show a higher rate of melting at their RBS compared with the mean of non-thermometers, contrary to our expectations these higher rates are not significant. Surprisingly, we also do not find any significant differences between rpoH thermometers from other -proteobacteria and E. coli non-thermometers.     

Ligand-dependent folding of the three-way junction in the purine riboswitch

Riboswitches are highly structured cis-acting elements located in the 5'-untranslated region of messenger RNAs that directly bind small molecule metabolites to regulate gene expression. Structural and biochemical studies have revealed riboswitches experience significant ligand-dependent conformational changes that are coupled to regulation. To monitor the coupling of ligand binding and RNA folding within the aptamer domain of the purine riboswitch, we have chemically probed the RNA with N-methylisatoic anhydride (NMIA) over a broad temperature range. Analysis of the temperature-dependent reactivity of the RNA in the presence and absence of hypoxanthine reveals that a limited set of nucleotides within the binding pocket change their conformation in response to ligand binding. Our data demonstrate that a distal loop-loop interaction serves to restrict the conformational freedom of a significant portion of the three-way junction, thereby promoting ligand binding under physiological conditions.     

Direct observation of the temperature-induced melting process of the Salmonella fourU RNA thermometer at base-pair resolution

In prokaryotes, RNA thermometers regulate a number of heat shock and virulence genes. These temperature sensitive RNA elements are usually located in the 5′-untranslated regions of the regulated genes. They repress translation initiation by base pairing to the Shine–Dalgarno sequence at low temperatures. We investigated the thermodynamic stability of the temperature labile hairpin 2 of the Salmonella fourU RNA thermometer over a broad temperature range and determined free energy, enthalpy and entropy values for the base-pair opening of individual nucleobases by measuring the temperature dependence of the imino proton exchange rates via NMR spectroscopy. Exchange rates were analyzed for the wild-type (wt) RNA and the A8C mutant. The wt RNA was found to be stabilized by the extraordinarily stable G14–C25 base pair. The mismatch base pair in the wt RNA thermometer (A8–G31) is responsible for the smaller cooperativity of the unfolding transition in the wt RNA. Enthalpy and entropy values for the base-pair opening events exhibit linear correlation for both RNAs. The slopes of these correlations coincide with the melting points of the RNAs determined by CD spectroscopy. RNA unfolding occurs at a temperature where all nucleobases have equal thermodynamic stabilities. Our results are in agreement with a consecutive zipper-type unfolding mechanism in which the stacking interaction is responsible for the observed cooperativity. Furthermore, remote effects of the A8C mutation affecting the stability of nucleobase G14 could be identified. According to our analysis we deduce that this effect is most probably transduced via the hydration shell of the RNA.     

The RNA chaperone StpA enables fast RNA refolding by destabilization of mutually exclusive base pairs within competing secondary structure elements

In bacteria RNA gene regulatory elements refold dependent on environmental clues between two or more long-lived conformational states each associated with a distinct regulatory state. The refolding kinetics are strongly temperature-dependent and especially at lower temperatures they reach timescales that are biologically not accessible. To overcome this problem, RNA chaperones have evolved. However, the precise molecular mechanism of how these proteins accelerate RNA refolding reactions remains enigmatic. Here we show how the RNA chaperone StpA of Escherichia coli leads to an acceleration of a bistable RNA’s refolding kinetics through the selective destabilization of key base pairing interactions. We find in laser assisted real-time NMR experiments on photocaged bistable RNAs that the RNA chaperone leads to a two-fold increase in refolding rates at low temperatures due to reduced stability of ground state conformations. Further, we can show that upon interaction with StpA, base pairing interactions in the bistable RNA are modulated to favor refolding through the dominant pseudoknotted transition pathway. Our results shed light on the molecular mechanism of the interaction between RNA chaperones and bistable RNAs and are the first step into a functional classification of chaperones dependent on their biophysical mode of operation.     

Clinical thermometry: possible causes and potential solutions to electronic,digital thermometer inaccuracies

Previous research has indicated that the improvements in safety, speed, and ease of use of the newer electronic, digital oral and tympanic clinical thermometers have been offset by a loss in accuracy and reliability when compared with the traditional glass/mercury oral clinical thermometers. The purpose of this study was to determine possible causes of the relative lack of accuracy and reliability in these thermometers and suggest logical potential solutions. Evidence has been reviewed, which suggests that problems with the tympanic thermometers are the result of the range of normal anatomical variability. The combination of an otoscope with the tympanic thermometers is suggested and discussed as a potential solution. Evidence has been reviewed, which suggests that problems with the newer oral thermometers are the results of the size of the temperature sensors and the speed with which they respond. The small sensor size results in variability in placement relative to the primary heat source. The speed of sensor response results in too great a sensitivity to local, acute vasomotor activity (clinical temperature noise). Potential solutions, including an increase in sensor size and a reduction in response speed are discussed.