**1. Introduction**

Whether it is within a host or within the non-host environment, bacteria experience frequent, and often extreme, changes within their immediate environment. In order to survive and thrive under different environmental conditions, bacteria have evolved various systems that function to sense changes in environmental conditions and mediate rapid adaptation in response to the

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specific change. One condition that varies between the different environments encountered by pathogenic and non-pathogenic bacteria alike is temperature. Environmental temperature has direct effects on several fundamental biological processes, including proper folding of proteins and optimum activity of enzymes. To counteract the potentially detrimental effects of altered temperature, bacteria have evolved several strategies to respond to changes in environmental temperature, including specific heat shock and cold shock responses. Moreover, for pathogenic bacteria, a change of environmental temperature is a critical cue that can indicate entry into the host and/or progression of the disease process within an infected host. In order to establish and progress an infection, bacteria not only need to efficiently adapt to changing environ‐ mental conditions but also need to precisely regulate the production of specific virulence factors — processes that are dependent on the ability of bacteria to sense specific changes in environmental conditions, including temperature.

One method of sensing alterations in environmental temperature is through changes in the secondary structure of RNA molecules. Double-stranded regions within a given RNA molecule tend to dissociate into single-stranded structures with an increase in environmental temperature. The temperature at which half of the population of a given double-stranded RNA molecule is in the single-stranded conformation is defined as the Tm, a feature that is com‐ monly used as a measurement for the stability of a given structure within an RNA molecule [1]. Due to its propensity to change conformation, an RNA structure that has a relatively low Tm is more responsive to changes in environmental temperature, a feature that facilitates its potential to act as a molecular thermosensors [2].

It is well established that translational efficiency is affected by the secondary structure of an RNA transcript, particularly that of the region containing the ribosome-binding site and/or start codon [3]. It was not until 1989, however, that the first *cis-*encoded temperature-respon‐ sive RNA regulatory element was identified [4]. Since that time, the rate at which temperatureresponsive *cis*-encoded regulatory RNA elements have been identified, and the concurrent understanding of how they function to control target gene expression has grown exponentially — a statement that is particularly true of temperature-sensing RNA regulatory elements in bacteria. Based on their innate responsiveness to changes in environmental temperature, regulatory RNA elements that function to modulate the translational efficiency for the transcript in which they are housed in response to alterations in temperature have been termed "RNA thermometers" (RNATs) [5]. Unlike metabolites-binding riboswitches, the activity of RNAT is not modulated by the absence or presence of a ligand [6,7]. The regulatory function of an RNAT relies solely on its innate chemical nature, which dictates the differential stability of a specific inhibitory structure at different environmental temperatures.

With the ever-increasing number of characterized RNATs, variability within this class of regulators is now coming to light. While the majority of RNATs are composed of sequences within the 5′ untranslated region (5′ UTR) of the regulated gene, some have now been shown to be composed, at least in part, of sequences within the coding region of the regulated transcript or by sequences within the coding region of a preceding gene within a polycistronic transcript [8,9]. In addition, the number of stem loops composing different RNATs varies, ranging from one in the simplest RNATs to five in the most complex RNATs [10,11]. Despite the variability among RNATs, they all share several basic fundamental features. Identifying and understanding the functional contribution of features conserved among characterized RNATs, as well as those that vary among this class of regulators, has and will continue to inform the foundational knowledge of the biological functions and chemical nature of these ubiquitous regulators. This chapter focuses on bacterial RNATs and provides a comprehensive summary of the current state of knowledge of RNATs, with emphasis given to discussions of the molecular mechanism underlying RNAT function, experimental techniques used to identify and characterize RNATs, families of bacterial RNATs, as well as the biological processes controlled by RNATs, and future directions of the field.

specific change. One condition that varies between the different environments encountered by pathogenic and non-pathogenic bacteria alike is temperature. Environmental temperature has direct effects on several fundamental biological processes, including proper folding of proteins and optimum activity of enzymes. To counteract the potentially detrimental effects of altered temperature, bacteria have evolved several strategies to respond to changes in environmental temperature, including specific heat shock and cold shock responses. Moreover, for pathogenic bacteria, a change of environmental temperature is a critical cue that can indicate entry into the host and/or progression of the disease process within an infected host. In order to establish and progress an infection, bacteria not only need to efficiently adapt to changing environ‐ mental conditions but also need to precisely regulate the production of specific virulence factors — processes that are dependent on the ability of bacteria to sense specific changes in

One method of sensing alterations in environmental temperature is through changes in the secondary structure of RNA molecules. Double-stranded regions within a given RNA molecule tend to dissociate into single-stranded structures with an increase in environmental temperature. The temperature at which half of the population of a given double-stranded RNA molecule is in the single-stranded conformation is defined as the Tm, a feature that is com‐ monly used as a measurement for the stability of a given structure within an RNA molecule [1]. Due to its propensity to change conformation, an RNA structure that has a relatively low Tm is more responsive to changes in environmental temperature, a feature that facilitates its

It is well established that translational efficiency is affected by the secondary structure of an RNA transcript, particularly that of the region containing the ribosome-binding site and/or start codon [3]. It was not until 1989, however, that the first *cis-*encoded temperature-respon‐ sive RNA regulatory element was identified [4]. Since that time, the rate at which temperatureresponsive *cis*-encoded regulatory RNA elements have been identified, and the concurrent understanding of how they function to control target gene expression has grown exponentially — a statement that is particularly true of temperature-sensing RNA regulatory elements in bacteria. Based on their innate responsiveness to changes in environmental temperature, regulatory RNA elements that function to modulate the translational efficiency for the transcript in which they are housed in response to alterations in temperature have been termed "RNA thermometers" (RNATs) [5]. Unlike metabolites-binding riboswitches, the activity of RNAT is not modulated by the absence or presence of a ligand [6,7]. The regulatory function of an RNAT relies solely on its innate chemical nature, which dictates the differential stability

With the ever-increasing number of characterized RNATs, variability within this class of regulators is now coming to light. While the majority of RNATs are composed of sequences within the 5′ untranslated region (5′ UTR) of the regulated gene, some have now been shown to be composed, at least in part, of sequences within the coding region of the regulated transcript or by sequences within the coding region of a preceding gene within a polycistronic transcript [8,9]. In addition, the number of stem loops composing different RNATs varies, ranging from one in the simplest RNATs to five in the most complex RNATs [10,11]. Despite the variability among RNATs, they all share several basic fundamental features. Identifying

of a specific inhibitory structure at different environmental temperatures.

environmental conditions, including temperature.

158 Nucleic Acids - From Basic Aspects to Laboratory Tools

potential to act as a molecular thermosensors [2].
