Regulating with RNA in Bacteria and Archaea

It may seem surprising that in almost all known life-forms, information-encoding transcripts are actively annihilated. Although at first glance this seems to be a potential waste of resources and loss of information, the anticipated advantages of restricting transcript lifetimes include fast response rates and a capacity to rapidly redirect gene expression pathways. In this way, destroying individual transcripts in a modulated manner might effectively enhance the collective information capacity of the living system. Escherichia coli has proven to be a useful model system to study such processes, and nearly 45 years ago, a hypothetical endoribonuclease was proposed by Apirion as the key missing factor that might account for the observed degradation patterns of mRNA in that bacterium. At the time this hypothesis was formulated, transcript decay in E. coli was best described as a series of endonucleolytic cleavages and subsequent fragment scavenging by 3′ exonucleases ( 1 ). A few years later, Apirion and colleagues reported the discovery of the endoribonuclease RNase E and showed it to be involved in processing of rRNA precursors ( 2 – 4 ), and the enzyme was subsequently discovered to also cleave an mRNA from T4 phage into a stable intermediate ( 5 ). Thus, RNase E seemed to be an ideal candidate for the proposed endonuclease factor to initiate RNA decay in bacteria. What made these findings surprising was that it had previously been thought that RNases might be specialized, with one set presumed responsible for mRNA decay and another set dedicated to stable RNA processing, whereas RNase E could perform both of these distinct tasks ( 6 ). This broad functionality has been found to be a recurrent feature of other RNases in E. coli and evolutionarily distant bacteria ( 7 ).

All living organisms, including the Gram-negative bacteria, have two major classes of RNA molecules. mRNAs contain the information for the synthesis of the various proteins that are required for a living cell. The so-called nontranslated RNAs, which include tRNAs, rRNAs, and small regulatory RNAs (sRNAs), provide the RNA components for ribosome assembly, protein synthesis, and the regulation of mRNA functionality based on RNA/RNA interactions. The highly diverse functions that these RNAs perform within the cell are possible due to numerous enzymes that are involved in posttranscriptional RNA metabolism. However, many of these enzymes have overlapping activities. Besides the normal cellular complement of enzymes that carry out the above functions, there are ribonucleases that are specifically associated with particular stress conditions as part of toxin/antitoxin (TA) systems.

Posttranscriptional regulation is a key modulator of gene expression in bacteria and allows their rapid adaptation to the environment. This regulation can be performed by proteins and/or RNA, by modifying either mRNA stability and/or translation. RNases are key enzymes in these processes. There are two main classes of RNases: endoribonucleases, which cleave directly in the “body” of the RNA; and exoribonucleases, which attack RNA from either its 5′ or 3′ end. Although RNases play a central role in RNA metabolism, these enzymes are not identical in Gram-negative and Gram-positive bacteria. For example, endoribonuclease E (RNase E) initiates bulk mRNA degradation and is essential in Escherichia coli, but this RNase is absent in many Firmicutes, such as Bacillus subtilis.

Temperature is an environmental cue that affects essentially every cellular process. To cope with sudden temperature changes, all living cells closely survey their ambient temperature through numerous sensory mechanisms, which involve regulatory proteins, changes in membrane fluidity, and impacts on DNA topology and RNA structures ( 1 , 2 ). Most of these mechanisms were initially discovered in studies of the heat shock response, which protects the cell from serious damage after a drastic shift to high temperatures. However, it is now established that subtle temperature changes already induce cellular responses. One process that involves reversible temperature changes is the entry and exit of mammalian pathogens into and from the host. A temperature of ∼37°C serves as a very good indicator to the bacterium that it is in a mammalian host. Various mechanisms regulating gene expression in response to host body temperature have been discovered, with some involving regulatory proteins and others utilizing sensory and regulatory RNAs. In this review, the main focus will be on RNA-mediated mechanisms; however, when the regulation involves a multicomponent regulatory network, protein-dependent regulatory events will be discussed.

Traditionally, the functional role of RNA was thought to be restricted to transferring genetic information from DNA to protein. However, the discovery of RNA elements mediating gene control, chemical reaction catalysis, and signal transduction has changed this perception fundamentally. Its ability to form complex three-dimensional structures that precisely present chemical moieties is imperative in enabling RNA to function as a biological catalyst, regulator, or structural scaffold.

Bacteria have evolved a wide array of mechanisms to control gene expression in response to environmental changes. These regulatory mechanisms ensure that specific genes are expressed under the appropriate physiological conditions, and they regulate every step of expression from transcription initiation to posttranslational modification and protein stability. The discovery of the T-box mechanism revealed that an uncharged tRNA can interact with an mRNA to regulate expression of the downstream coding region ( 1 ) ( Fig. 1 ). This mechanism was the first of many regulatory systems to be discovered in which cis-encoded RNA responds directly to a physiological signal to control gene expression through structural rearrangements. Regulatory RNAs of this type, termed riboswitches, have become an intense focus of research, and to date dozens of riboswitch classes that respond to various signals have been identified and characterized, including those that respond to temperature, pH, and metabolites such as enzyme cofactors, amino acids, and nucleotides ( 2 ).

Despite the many roles for RNA as a regulator in eukaryotes, archaea, and bacteria, the rRNA is the most abundant cellular RNA and the size of the rRNA outstrips nearly all other functional RNAs. Furthermore, the ribosome is also composed of >50 ribosomal proteins (r-proteins), the majority of which directly contact the rRNA, forming specific interactions with RNA ( 1 ). Since most regulatory RNAs in bacteria appear to be relatively recent inventions ( 2 – 5 ), they most certainly have evolved in the context of abundant rRNA and r-proteins, and thus have been shaped by them. Many regulatory RNA structures contain portions that bear strong resemblance to motifs within the rRNA. Some of this similarity is due to the role that rRNA plays in our understanding of RNA structure, and in other cases it is due to interaction with an r-protein. This review will first illustrate the role of the ribosome in our understanding of RNA structures generally and subsequently examine how r-proteins may interact with RNA outside the ribosome to act in a regulatory capacity.

An extraordinarily diverse range of genetic regulatory mechanisms has been discovered in the half century since Francois Jacob and Jacques Monod first proposed the operon model of gene regulation ( 1 ). Studies based on this model identified a soluble regulator, located distally from the targeted operon, that acts to repress transcription initiation of the lac operon. This discovery led to the identification and characterization of many more repressor proteins, each acting in modestly different ways to reduce the efficiency of transcription initiation. Soon followed discoveries of other types of transcriptional regulators, including those that activate gene expression by enhancing transcription initiation. And now, in an era in which bacterial genome sequences can be acquired and draft-annotated in mere days and at low cost, it is clear that all bacteria encode dozens or hundreds of proteins that regulate transcription initiation and that this “layer” of genetic regulation is both ubiquitous and profoundly important. However, perhaps because transcription initiation is so universally recognized as a key point of regulatory influence ( 2 ), later stages of transcription elongation have not yet been sufficiently analyzed for genetic regulation. While the molecular mechanisms of transcription have been, and continue to be, intensively investigated, the biological extent of postinitiation regulatory mechanisms has been incompletely analyzed. Transcription initiation is only the first stage of gene expression. The stages that follow include transcription elongation, transcription termination, translation, and mRNA degradation; each of these stages can be subjected to genetic regulatory control ( 3 ).

The most common result of the translation of a gene is the production of a single protein product ( Fig. 1a ). However, the redundancy of the genetic code and the plasticity of the mRNA structure allow for expansions of the proteome by unorthodox interpreting of genetic information. A familiar strategy leading to unusual interpretation of genetic information is collectively known as recoding and has been discussed in several excellent reviews ( 1 – 4 ). The most conventional recoding involves programmed ribosomal frameshifting and can generate two gene products that are identical in their N-terminal segments but differ in the sequences of their C termini ( Fig. 1b ).

In bacteria and archaea, translation initiates with a 30S ribosomal subunit interacting with an initiator tRNA at the ribosome binding site on a canonical mRNA to form a stable translation initiation complex that is primed for elongation. Canonical mRNAs contain both 5′ and 3′ untranslated regions (UTRs) containing information that will influence the stability and translation efficiency of the mRNA. Within the 5′ UTR, these signals can include ribosome recognition regions such as purine-rich Shine-Dalgarno (SD) sequences that are complementary to the anti-SD (aSD) sequence near the 16S rRNA 3′ terminus ( 1 ), AU-rich sequences that interact with ribosomal protein (r-protein) bS1 ( 2 , 3 ) and prevent the formation of secondary structures, and enhancer regions. Additionally, 5′ UTRs may contain sequences that can be bound by trans-acting elements (i.e., proteins, antisense and small regulatory RNAs, or low-molecular-weight effectors) to change secondary structures or block translation initiation regions. Therefore, the regulatory and translation initiation signals are primarily contained within the 5′ UTR. Despite this functional importance of the 5′ UTR, there exists a class of mRNAs that are completely devoid of 5′ UTRs or possess very short 5′ UTRs. These mRNAs lack the SD sequence and any other translational signals and are so named leaderless mRNAs (lmRNAs). Thus, the mechanism underlying their recognition and binding by the translational apparatus is still not entirely elucidated.

Toxin-antitoxin (TA) systems are, by definition, simple genetic loci composed of two genes: a toxin and an antitoxin that counteracts either the toxin’s action or its expression. Usually, toxin synthesis leads to growth arrest or death of the bacterium that produces it by inhibiting essential cellular processes such as replication, translation, or cell division ( 1 ). Analogies can be made with other gene pairs with a similar bicistronic operon organization, such as bacteriocins, restriction-modification systems, and type VI secretion system effector/immunity proteins ( 2 ). For instance, they are often found within mobile genetic elements and pathogenicity islands. However, canonical TA systems generally act on the cell that produces them, and in contrast to bacteriocins, they are not involved in interbacterial competition. Thus, the presence of TA-encoded toxins in bacterial genomes strongly depends on the expression of their cognate antitoxins ( 3 ). TA systems can be classified into six different types depending on the nature and mode of action of the antitoxin (for a review, see reference 4 ). While the toxins are always proteins, antitoxins can be either proteins (types II, IV, V, and VI) or small RNAs (sRNAs, types I and III TA systems). Antitoxins can act by (i) sequestering the toxin (types II and III), (ii) inhibiting its expression (types I and V), or (iii) counterbalancing its activity (type IV). In this review, we will focus on type I TA systems, in which an antisense RNA plays the role of antitoxin to prevent the synthesis of its cognate toxin by directly base-pairing to the mRNA.

The first documented cis-encoded antisense RNAs (asRNAs) in bacteria were the RNA I, controlling ColE1 replication ( 1 ), and the OOP asRNA of bacteriophage λ ( 2 – 4 ). However, until the year 2007 a mere ∼10 bacterial asRNAs had been characterized ( 5 ). It was only with the advent of global approaches for the analysis of bacterial transcriptomes that it was recognized that actually a substantial fraction of transcripts, in fact, constitute asRNAs. The first hints obtained with high-density microarrays pointed at antisense transcription linked to possibly as many as 3,000 to 4,000 open reading frames in Escherichia coli ( 6 ), more recently reinforced by the finding that asRNAs originate from 37% of all transcription start sites (TSSs) ( 7 ), which might still be an underestimation of the initial level of antisense transcription ( 8 ). By the hybridization of directly labeled RNA instead of cDNA to high-density microarrays, a high number of strongly expressed asRNAs were experimentally identified in the model cyanobacterium Synechocystis sp. PCC 6803 ( 9 ). The direct labeling of RNA avoided the artificial second-strand synthesis in the production of cDNA, a step at which experimental artifacts might be introduced ( 10 ). In agreement with the initial evidence, numerous transcriptome studies have demonstrated more recently that a substantial fraction of the discovered TSS in vastly different bacterial taxa is not associated with a protein-coding gene. Internal parts of coding regions in sense and antisense orientation are massively transcribed, a phenomenon often referred to as pervasive transcription ( 11 , 12 ).

One major paradigm for RNA-based regulation in both eukaryotes and prokaryotes is small regulatory RNAs (sRNAs) that pair with mRNAs, leading to changes in translation and mRNA stability. In bacteria, rather than the highly processed very short microRNAs found in eukaryotes, these sRNAs are generally on the order of 50 to 200 nucleotides (nt) long, and in the Gram-negative organisms that are the major focus of this review, annealing of sRNAs to their target mRNAs is usually dependent on the RNA chaperone, Hfq. Annealing can lead to positive regulation of translation, by remodeling inhibitory RNA structures or blocking access of negative regulators (for instance, RNases or the Rho transcription termination factor), or negative regulation, by inhibiting translation, recruiting RNases, or both. A given sRNA can have multiple targets, and can carry out both negative and positive regulation ( 1 – 3 ).

Carbohydrates are degraded in central metabolic pathways, namely, glycolysis, the pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle, to fuel cells with energy and building blocks to synthesize all biomolecules. A functional carbohydrate metabolism requires sufficient supply with carbon sources but also coordination with the availability of other nutrients and cellular activities. Hence, bacterial carbohydrate metabolism is controlled at all levels by large and densely interconnected regulatory networks ( 1 ). In recent years, posttranscriptional mechanisms involving small regulatory RNAs (sRNAs) have emerged as an additional layer in these networks. Extensive cross talk of sRNAs with transcriptional regulators ensures a fine-tuned and coordinated metabolism.

Global metabolic regulatory networks allow microorganisms to survive periods of nutrient starvation or stress resulting from changes in the environment. Besides the metabolic regulatory network controlling uptake and metabolism of carbon and energy sources, the respective regulatory system responsible for acquisition of different nitrogen sources is highly important for surviving nutrient starvation. Bacteria and archaea developed several strategies for uptake and utilization of various nitrogen sources, like ammonium, amino acids, and inorganic nitrogen compounds. In the absence of any available nitrogen source, nitrogen-fixing microorganisms—so-called diazotrophs—are able to use molecular nitrogen (N2), a process exclusively limited to prokaryotes but found in both domains, Bacteria and Archaea (for reviews, see references 1 – 5 ). Uptake and utilization of alternative nitrogen sources is strictly regulated due to higher energy demands, and respective enzymes are thus tightly regulated in response to the available nitrogen source to minimize energy costs for the nitrogen metabolism. The well-studied and -documented regulation occurs mainly at the transcriptional and posttranslational level, where the molecular mechanisms differ from microorganism to microorganism as well as between bacteria and archaea (e.g., see references 6 – 9 ). In bacteria, the transcription of nitrogen-relevant components is generally regulated by major global transcriptional regulators, most of which are constitutively expressed ( 9 ). The two-component regulatory system NtrB/NtrC—present in numerous proteobacteria—regulates the transcriptional activation of σ54 promoters recognized by the alternative RNA polymerase (RNAP) containing the sigma factor σ54 (RpoN) ( 8 ). In cyanobacteria, transcriptional regulation of central nitrogen metabolism is mediated by the master regulator NtcA, a transcription factor belonging to the CRP (cyclic AMP receptor protein) family. NtcA forms dimers capable of binding to its specific promoter motifs in a nitrogen-dependent way, and thus coordinates the cellular response to nitrogen availability at the transcriptional level (reviewed in references 10 and 11 ).

Iron is one of the most abundant elements on earth. Due to its chemical properties, in particular its redox potential, it was used as a cofactor in a large number of proteins since the emergence of life. Before the appearance of an oxidative atmosphere, iron was found mainly in its reduced, ferrous form (Fe2+). Fe2+ is typically the bioreactive form of iron that is found in proteins, as an isolated ion, in the center of porphyrin to form heme, or in coordination with sulfur atoms to constitute so-called Fe-S cluster cofactors ( 1 – 3 ). Bacteria contain many iron-using proteins involved in a plethora of reactions, mainly, but not limited to, aerobic and anaerobic respiration, the tricarboxylic acid (TCA) cycle, photosynthesis, N2 fixation, and DNA biosynthesis.

Bacteria use the production, release, and detection of extracellular chemical signaling molecules called autoinducers (AIs) to regulate gene expression at the population level, a widespread phenomenon known as quorum sensing (QS). The first reviews of small RNA (sRNA) control of QS were published in 2006 and 2007 ( 1 , 2 ). At that time, two QS systems had been described that are entirely dependent on sRNA-based regulation, namely, the QS circuits of the Gram-negative family Vibrionaceae and the agr QS circuit of the Gram-positive staphylococci. A decade later, these two examples have become paradigms of sRNA-based regulation, and have taught us important regulatory principles relevant not only to QS but to bacterial sRNA-based regulation in general. I begin this review by describing the progress made in understanding sRNA-based execution of the QS response in these two systems. In many other bacteria the core executors of QS are not sRNAs, but sRNAs are nevertheless identified as auxiliary regulators that directly or indirectly affect components of the QS machinery. In particular, sRNA-based regulation of the production of enzymes that catalyze AI synthesis appears as a common trend across numerous unrelated QS circuits, and such examples will be highlighted. Due to space limitations, this review is solely focused on riboregulation carried out by small trans-acting RNA molecules that are encoded at loci distinct from those of their target(s), thereby excluding cis-acting regulatory RNA elements such as riboswitches and attenuators, as well as antisense RNA transcripts. Notably, QS has only been sparsely connected to cis-acting riboregulation in the literature thus far ( 3 , 4 ).

Infectious diseases caused by bacterial pathogens still constitute one of the major human health threats ( 1 ). Within the host body, pathogenic bacteria face a wide variety of hostile microenvironments and encounter numerous different host cell types as well as commensal bacteria of the resident microbiota. To cope with these environmental changes and respond adequately to any interacting cell, bacterial pathogens have to tightly control their gene expression during infection, in part by means of regulatory RNAs.

The Csr (carbon storage regulator) or Rsm (repressor of stationary-phase metabolites) system is among the most extensively studied bacterial RNA-based regulatory systems. Its central component, the RNA binding protein CsrA (RsmA), was uncovered by a transposon mutagenesis screen designed to identify regulators of gene expression in the stationary phase of growth, using glycogen biosynthesis and glgC’-‘lacZ expression as reporters ( 1 ). Understanding of RNA binding proteins and their roles in regulation was limited at that time, but included Hfq and ribosomal proteins that mediate negative feedback by binding to their mRNAs ( 2 – 4 ). Soon after its discovery, the regulatory role of CsrA began to emerge, which included repression of other genes similar to glgC, which are expressed in stationary phase or under stress conditions ( 5 ), and evidence that CsrA activates gene expression that supports robust growth ( 6 ). Discoveries that CsrA (RsmA) regulates virulence genes of pathogens associated with plant disease ( 7 ) and mammalian cell invasion ( 8 ) offered early glimpses of the widespread roles played by CsrA proteins in microbe-host interactions ( 9 ). The role of CsrA in biofilm formation ( 10 – 14 ), quorum sensing ( 15 ), carbon metabolism ( 6 , 16 , 17 ), motility ( 18 , 19 ), and stress responses ( 14 , 20 – 23 ) is now well documented in Escherichia coli and other species. New functions of CsrA are being uncovered at a rapid pace through the use of transcriptomics, proteomics, metabolomics, and other systems approaches ( 12 , 13 , 20 , 24 – 35 ).

It is now well established that small RNAs (sRNAs) have diverse and widespread roles in regulating gene expression in all organisms ( 1 – 4 ). Mechanisms of action are varied but can be broadly classified into three categories: (i) sRNAs that act by base-pairing to target RNAs; (ii) sRNAs that act to modulate protein activity through direct RNA-protein interaction; and (iii) sRNAs that have intrinsic function (e.g., catalytic). Two well-studied sRNA families that modulate protein activity include sRNAs that regulate CsrA protein and 6S RNA, which regulates RNA polymerase (RNAP) and is the focus here as well as in several other reviews ( 5 – 8 ). 6S RNA was first identified in Escherichia coli ( 9 , 10 ), which remains the best-understood model of 6S RNA function, although identification of 6S RNAs and their roles in diverse bacterial species has been an active area of research in the past decade. Here, information for E. coli will be presented first, followed by discussion of similarities and differences known or postulated for 6S RNAs in diverse species.

In contrast to most bacterial noncoding RNAs (ncRNAs) ( 1 , 2 ), Y RNAs were initially characterized in human cells and only later shown to exist in bacteria. The human RNAs were discovered because they are found complexed with the Ro 60-kDa autoantigen (Ro60), a ring-shaped protein that is a clinically important target of autoantibodies in patients with two systemic autoimmune rheumatic diseases, systemic lupus erythematosus and Sjögren’s syndrome ( 3 , 4 ). Y RNAs and their Ro60 protein partner were subsequently shown to be present in all examined animal cells as well as in a subset of bacteria ( 5 – 13 ). The number of distinct Y RNAs varies between species, with most characterized organisms having between two and four ( 4 , 5 , 8 , 12 , 13 ). Although all experimentally verified Y RNAs are between 69 and 150 nucleotides, homology searches predict that some bacterial Y RNAs may exceed 200 nucleotides ( 13 ).

Noncoding RNA sequences fold into useful structures that regulate gene expression as ribozymes, metabolite-binding sensors, or antisense RNAs ( 1 – 5 ). These regulatory RNAs are chaperoned by diverse families of RNA-binding proteins, and the loss of RNA chaperone proteins can lead to impaired growth, reduced tolerance to stress, and reduced virulence ( 6 – 11 ). RNA chaperones also facilitate conformational rearrangements during ribosome biogenesis ( 12 ) and eukaryotic pre-mRNA splicing ( 13 ).

Today, RNA is no longer seen as a mere intermediary, transferring information from genes to proteins. It has become more and more obvious that RNA plays additional biological roles in living organisms ( 1 , 2 ). Such roles are highly diverse, including catalytic and gene-regulatory functions. While the catalytic roles in present-day biology appear limited, regulatory RNAs are abundant in all kingdoms of life and control central biological processes from cell cycle progression, differentiation, adaptation, and stress response to pathological processes, such as carcinogenesis or inflammation ( 3 , 4 ).

Spatial and temporal localization of macromolecules, including RNAs, reflects the compartmentalization of living cells and plays important roles in gene expression and regulation. In eukaryotic cells, physical separation between the transcription and translation machineries in the nucleus and cytoplasm, respectively, naturally results in the synthesis, processing, and translation of mRNA to be spatially disconnected. Both mRNA localization and localized translation can be important regulatory mechanisms underlying embryonic patterning, asymmetric cell division, epithelial polarity, cell migration, and neuronal morphogenesis ( 1 , 2 ). RNAs can be transported in the eukaryotic cell in several ways, such as (i) vectorial movement of mRNA by direct coupling to motor proteins, (ii) transport of mRNA by hitchhiking on another cargo, (iii) random transport of mRNA-motor complexes and local enrichment of mRNAs at target sites, or (iv) diffusion and motor-driven cytoplasmic flows with subsequent localized anchorage of the mRNA ( 3 ). Moreover, localized translation induction by phosphorylation and activation of translation initiation factors and their regulators in response to localized signals have been reported to impact gene regulation in eukaryotes ( 4 ).

MicroRNAs (miRNAs) are 20- to-24-nucleotide (nt)-long RNAs that guide Argonaute proteins to silence mRNA expression in animal and plant cells ( 1 – 3 ). Similarly to bacterial trans-encoded small RNAs (sRNAs), miRNAs act by establishing imperfect base-pair interactions with seed sequences that can be as short as 6 to 8 nt. Seeking ways to selectively control miRNA activity in vivo, a decade ago Ebert and coworkers engineered transcripts containing multiple tandemly arranged target sites for one or more miRNAs and had these constructs expressed at high levels in transfected mammalian cells ( 4 ). They found the exogenous RNAs to have the ability to sequester (“soak up”) the miRNAs, relieving the regulation of their natural targets. The authors termed the artificial transcripts “microRNA sponges.” At about the same time, a study on the mechanism responsible for inhibiting the activity of a miRNA (miR399) in plant cells identified an endogenous noncoding RNA, named IPS1, that could base-pair with miR399 and compete for its binding to the primary target ( 5 ). This indicated that a natural RNA could have sponge-like activity and that target site amplification was not required for this effect. Following these initial findings, several examples of miRNA target mimicry have been described involving different types of coding and noncoding RNAs ( 6 , 7 ), including some of viral origin ( 8 , 9 ). Particularly noteworthy is the case of the circular antisense RNA named CDR1as, highly expressed in human and mouse brain, which harbors as many as 74 potential target sites for the miR-7 miRNA and thus closely fulfills the original definition of a sponge ( 10 ). Recent evidence showed CDR1as to be a highly efficient miR-7 sponge in vivo: in cells lacking CDR1as, deregulation of miR-7 networks leads to profound defects in brain development and function ( 11 ).

Regulatory RNAs have emerged as important regulators of gene expression in all kingdoms of life, and many advances toward the understanding of their biology have been achieved in bacteria. Bacterial regulatory RNAs are often also referred to as small RNAs (sRNAs), as most of them range in size from 50 to 400 nucleotides. While it was recognized early on that these sRNAs can ensure extremely diverse biological functions, such as trans-translation (transfer-messenger RNA), ribonucleolytic activity (RNA moiety of the RNase P), or even involvement in protein secretion (4.5S RNA), the shared efforts of multiple groups in the last 2 decades have led to the identification of a plethora of sRNAs in virtually all bacteria. Many of these act as posttranscriptional regulators of gene expression and generally function by imperfectly base-pairing to target mRNA(s), leading to changes in their translation and/or stability.

Bacteria have evolved elaborate responses to sense, protect against, and help recovery from stressful fluctuations in environmental conditions. In the past decade, small regulatory RNAs (sRNAs) have emerged as important players in the posttranscriptional regulation of various stress responses. Advances in deep sequencing have led to the identification of hundreds of these sRNAs, which range from 50 to 350 nucleotides (nt) in length, thereby greatly increasing the numbers of known sRNAs ( 1 ). Usually, these sRNA regulators are thought to be noncoding and are generally presumed to act by modulating the stability and translation of mRNAs through short base-pairing interactions or by binding to and modulating the activities of RNA-binding proteins.

As our understanding of the transcriptional landscape of bacteria continues to expand, it has become clear that noncoding small RNAs (sRNAs) play a pivotal regulatory role ( 1 – 3 ). Typically 50 to 400 nucleotides in length, sRNAs posttranscriptionally regulate gene expression, usually by base-pairing with one or more mRNA targets ( 4 ). sRNAs likely provide certain advantages over protein regulators because they act quickly, are relatively metabolically inexpensive, and provide an additional way to respond to environmental signals ( 1 ). Beyond these basic characteristics, however, the roles of bacterial sRNAs are extremely diverse: they are capable of upregulating or downregulating translation, stabilizing mRNAs or targeting them for degradation, sharing targets, and/or targeting multiple mRNAs. Variability in their sequence, structure, and how and when they are transcribed allows them to meet a wide range of nuanced regulatory needs based on the diverse environments to which bacteria must adapt.

The impact that the study of phages, both in their lytic form and as prophages integrated into bacterial chromosomes, has had on molecular biology and microbiology is hard to overstate. The ease of phage manipulation helped establish several of the central dogmas in molecular biology. For example, characterization of various phage DNA polymerases contributed to the understanding of replication ( 1 , 2 ), and models of transcription regulation were greatly influenced by studies of cI, the phage λ repressor ( 3 , 4 ). Phages also have continually provided important tools such as transduction, the phage-assisted movement of DNA from one bacterium to another, which has been an essential tool since the early years of molecular biology ( 5 , 6 ). As another example, the development of chain termination DNA-sequencing approaches benefited from single-stranded DNA cloning vectors derived from phage M13 ( 7 ).

Although bacteria harbor far fewer long noncoding RNAs (ncRNAs) than eukaryotes, the known classes of large, structured ncRNAs in bacteria perform essential roles in the core processes of information transfer, metabolism, and physiological adaptation ( 1 ). For example, many classes are central to genetic information processing: rRNAs act as ribozymes ( 2 ) to translate mRNAs, RNase P ribozymes process precursor tRNAs ( 3 , 4 ), transfer-messenger RNAs (tmRNAs) rescue stalled ribosomes ( 5 , 6 ), and riboswitches bind ions and metabolites to regulate gene expression ( 7 – 10 ). Furthermore, most of the large, structured ncRNA classes whose functions are known operate as ribozymes that perform essential chemical reactions such as peptide bond formation ( 2 ), RNA splicing ( 11 , 12 ), and RNA cleavage ( 3 ). Two of these ribozyme classes, namely group I and group II introns, are sometimes components of selfish genetic elements that both splice mRNAs and mobilize to various regions in DNA genomes ( 13 , 14 ). Of course, many self-splicing ribozymes also carry protein-coding regions, located either in their exon flanks or inserted into noncritical portions of their ribozyme structure. However, these coding regions are usually incidental to the main functions performed by the ncRNA’s structure. Collectively, these ncRNAs have an enormous influence on both genetic and cellular processes, which suggests the intriguing possibility that newly found large ncRNA classes may also serve fundamental roles in biology.

RNAs have been known to perform a vast amount of regulatory functions in bacteria and archaea. Small RNAs (sRNAs) and riboswitches are two extensively studied classes of regulatory RNAs. sRNAs are trans-acting RNA elements between ∼50 and 500 nucleotides (nt) in length that are either independently transcribed or processed from a nontarget mRNA, and contain imperfect complementarity to the target mRNA to perform posttranscriptional regulatory functions. On the contrary, riboswitches are cis-regulatory structured RNA elements in the untranslated regions of mRNAs, capable of regulating downstream gene expression through small-molecule ligand-induced conformational switching. These regulatory RNAs have revealed the precise and sophisticated nature of natural gene regulatory networks and have inspired efforts to mimic these mechanisms and functions by engineering RNA tools for an increasing number of synthetic biology applications.

Transcriptional profiling is a valuable part of the functional genomics toolbox. Since the developments in nanotechnology and imaging that led to the invention of next-generation sequencing ( 1 ), study of the bacterial transcriptome at the level of the individual nucleotide has proved fruitful. Scientists are now generating increasing amounts of transcriptomic data that need to be managed, analyzed, and stored in an appropriate manner ( 2 ). The current need for systematic and accessible approaches for the analysis of gene expression has focused bioinformatic efforts into developing tools for processing transcriptomic data.

For over a decade, prokaryotic and eukaryotic RNA biology exploration has unveiled the multifaceted and central contribution of RNA-based control in all domains of life. RNA interactions are at the core of many regulative processes and have hence been heavily studied by wet-lab and biocomputational researchers alike. Within this review, we focus on biocomputational methods and outline the technical details of standard algorithms for RNA secondary structure and RNA-RNA interaction prediction. Furthermore, we highlight their application in the context of prokaryotic RNA biology.