**6. Future directions**

Although RNA-dependent regulation of gene expression has been a topic of active investiga‐ tion for decades, investigations of RNATs are much more recent, with less than 100 RNATs having been identified to date (Table 1). Of note, RNATs vary in key structural features and influence different essential physiological processes.



RNATs have also been implicated in controlling the expression of virulence-associated genes that encode factors involved in adhesion and immune evasion. For example, three virulenceassociated genes in *N. meningitis* have been found to be regulated by RNATs: *cssA,* a gene encoding a factor involved in capsule production; *fHbp,* a gene encoding a factor H binding protein; and *lst,* a gene encoding a factor required for modifications of lipopolysacccharides [24]. In *L. interrogans, ligA* and *ligB*, two genes encoding putative lipoprotein, are also regulated by RNATs [43]. Additionally *P. aeruginosa rhlA,* a gene encoding an enzyme required for the synthesis of rhamnolipid, a compound that can prevent killing of the bacteria by host immune system, is regulated by an RNAT [37]. Except for *rhlA* RNAT, which is a member of the RSOElike family, these other RNATs mentioned above have unique structures and thus are not

To date, two genes involved in the acquisition of essential nutrients have been shown to be regulated by RNATs: *S. dysenteriae shuA*, a gene encoding an outer membrane heme-binding protein, and its homologous gene *chuA* in pathogenic *E. coli* [11]. Translation of *shuA/chuA* is controlled by a FourU RNAT located in the relative large 5′ UTR of the corresponding gene. Production of ShuA or ChuA facilitates the utilization of iron from heme, a potential source of essential iron found only within the relatively warm environment of the infected host [54].

For many pathogenic bacteria, the transmission from one host to the next involves exposure to different environments with different temperatures. The expression of many virulenceassociated genes is influenced by environmental temperature, a signal that varies between the host and non-host environments. With an increasing number of virulence-associated genes that are now known to be regulated by the activity of RNATs, it is possible that temperaturedependent regulation mediated by RNATs will emerge as one of the basic regulatory strategies utilized by pathogenic bacteria. The full and potentially expansive role that RNATs play in

Although RNA-dependent regulation of gene expression has been a topic of active investiga‐ tion for decades, investigations of RNATs are much more recent, with less than 100 RNATs having been identified to date (Table 1). Of note, RNATs vary in key structural features and

*Agrobacterium tumefaciens hspAT1* & *hspAT2* Small heat shock protein Balsiger *et. al.*<sup>2004</sup>

*Bartonella henselae ibpA2* Small heat shock protein Waldminghaus *et. al.*

[33]

2005 [36]

**RNAT type Organism Gene Function of the gene Reference**

members of the ROSE-like or FourU families of regulators.

172 Nucleic Acids - From Basic Aspects to Laboratory Tools

controlling virulence of pathogenic bacteria is yet to be revealed.

influence different essential physiological processes.

**6. Future directions**

ROSE-element



**Table 1.** Summary of currently identified RNA thermometers

**RNAT type Organism Gene Function of the gene Reference**

*Pseudomonas aeruginosa rhlA*

*Salmonella enterica*

174 Nucleic Acids - From Basic Aspects to Laboratory Tools

*Escherichia coli* (some

*Vibrio cholerae toxT*

strains)

FourU element *Shewanella oneidensis ibpA* Small heat shock protein Waldminghaus *et. al.*

*Shigella flexneri ibpA* & *ibpB* Small heat shock protein Waldminghaus *et. al.*

*Sinorhizobium meliloti ibpA* & *b21295* Small heat shock protein Waldminghaus *et. al.*

*Vibrio cholerae hspA* Small heat shock protein Waldminghaus *et. al.*

*Vibrio parahaemolyticus hspA* Small heat shock protein Waldminghaus *et. al.*

*Vibrio vulnificus hspA* Small heat shock protein Waldminghaus *et. al.*

*Yersinia pestis ibpA* & *ibpB* Small heat shock protein Waldminghaus *et. al.*

*Escherichia coli htrA* Stress-responding periplasmic

*Shigella dysenteriae shuA* Outer membrane heme-binding

*Yersinia pestis lcrF* Transcriptional activator of

*Yersinia pseudotuberculosis virF (lcrF)* Transcriptional activator of

*htrAp3*

rhamnolipids

protease

Enzymes involved in the production of biosurfactant

*agsA* Small heat shock protein Waldminghaus *et. al.*

Stress-responding periplasmic protease (transcribed from the 3rd

promoter of the gene)

Transcriptional activator of virulence factors (including

multiple virulence genes

multiple virulence genes

*chuA* Outer membrane heme-binding protein

protein

cholera toxin)

2005 [36]

2005 [36]

2005 [36]

2005 [36]

2005 [36]

2005 [36]

2005 [36]

2014 [37]

2007 [22]

[10]

[10]

[40]

[42]

[41]

Grosso-Becerra *et. al.*

Klinkert et. al. 2012

Klinkert et. al. 2012

Kouse *et. al.* 2013 [11]

Kouse *et. al.* 2013 [11]

Weber et. al. 2014

Böhme *et. al.* 2012 [41]; Hoe *et. al.* 1993

Böhme *et. al.* 2012

Despite their differences, all currently characterized RNATs are thought to share the same basic zipper-like temperature-responsive molecular mechanism, based on which both experimental and therapeutic applications can be derived. For example, artificial RNATs that have only a single hairpin to perform the temperature-dependent inhibition of translation have now been designed [55]. These artificial RNATs can be used as genetic tools to manipulate target gene expression. In the aspect of applying knowledge of RNATs in developing thera‐ peutics, it is conceivable that compounds can be developed that would specifically stabilize the inhibitory structure within a given RNAT, thus decreasing expression of this target gene. Utilizing such an approach to inhibit the production of an essential gene product or virulence factor could prevent or limit infections by a variety of pathogenic bacteria.

Future applications of RNATs as genetic tools and/or drug targets are dependent on an increased understanding of these ubiquitous regulatory elements. With the maturation and development of experimental techniques, we could identify additional RNATs and study the molecular mechanisms underlying their regulatory activity in even greater detail. Moreover, due to the fundamental roles of RNA in the biological world, there is a great potential that RNATs also exist in archaea and eukaryotes. Further investigation and characterization of the conserved features and mechanisms of RNATs along with an understanding of the function of their regulatory targets could provide insight into the complex evolution of gene regulation. With the rate at which advances have been made in the field of RNA-mediated regulation, and specifically within the study of RNATs, there is no doubt that these and other important findings will be revealed sooner than later.
