Transcription Attenuation Mechanism Research Articles

NusG-dependent RNA polymerase pausing is a common feature of riboswitch regulatory mechanisms.

Transcription by RNA polymerase is punctuated by transient pausing events. Pausing provides time for RNA folding and binding of regulatory factors to the paused elongation complex. We previously identified 1600 NusG-dependent pauses throughout the Bacillus subtilis genome, with ∼20% localized to 5' leader regions, suggesting a regulatory role for these pauses. We examined pauses associated with known riboswitches to determine whether pausing is a common feature of these mechanisms. NusG-dependent pauses in the fmnP, tenA, mgtE, lysP and mtnK riboswitches were in strategic positions preceding the critical decision between the formation of alternative antiterminator or terminator structures, which is a critical feature of transcription attenuation mechanisms. In vitro transcription assays demonstrated that pausing increased the frequency of termination in the presence of the cognate ligand. NusG-dependent pausing also reduced the ligand concentration required for efficient termination. In vivo expression studies with transcriptional fusions confirmed that NusG-dependent pausing is a critical component of each riboswitch mechanism. Our results indicate that pausing enables cells to sense a broader range of ligand concentrations for fine-tuning riboswitch attenuation mechanisms.

A conserved uORF in the ilvBNC mRNA of Corynebacterium species regulates ilv operon expression.

Computational methods can be used to identify putative structured noncoding RNAs (ncRNAs) in bacteria, which can then be validated using various biochemical and genetic approaches. In a search for ncRNAs in Corynebacterium pseudotuberculosis, we observed a conserved region called the ilvB-II motif located upstream of the ilvB gene that is also present in other members of this genus. This gene codes for an enzyme involved in the production of branched-chain amino acids (BCAAs). The ilvB gene in some bacteria is regulated by members of a ppGpp-sensing riboswitch class, but previous and current data suggest that the ilvB-II motif regulates expression by a transcription attenuation mechanism involving protein translation from an upstream open reading frame (uORF or leader peptide). All representatives of this RNA motif carry a start codon positioned in-frame with a nearby stop codon, and the peptides resulting from translation of this uORF are enriched for BCAAs, suggesting that expression of the ilvB gene in the host cells is controlled by attenuation. Furthermore, recently discovered RNA motifs also associated with ilvB genes in other bacterial species appear to carry distinct uORFs, suggesting that transcription attenuation by uORF translation is a common mechanism for regulating ilvB genes.

Expression of Bacillus subtilis ABCF antibiotic resistance factor VmlR is regulated by RNA polymerase pausing, transcription attenuation, translation attenuation and (p)ppGpp.

Since antibiotic resistance is often associated with a fitness cost, bacteria employ multi-layered regulatory mechanisms to ensure that expression of resistance factors is restricted to times of antibiotic challenge. In Bacillus subtilis, the chromosomally-encoded ABCF ATPase VmlR confers resistance to pleuromutilin, lincosamide and type A streptogramin translation inhibitors. Here we show that vmlR expression is regulated by translation attenuation and transcription attenuation mechanisms. Antibiotic-induced ribosome stalling during translation of an upstream open reading frame in the vmlR leader region prevents formation of an anti-antiterminator structure, leading to the formation of an antiterminator structure that prevents intrinsic termination. Thus, transcription in the presence of antibiotic induces vmlR expression. We also show that NusG-dependent RNA polymerase pausing in the vmlR leader prevents leaky expression in the absence of antibiotic. Furthermore, we demonstrate that induction of VmlR expression by compromised protein synthesis does not require the ability of VmlR to rescue the translational defect, as exemplified by constitutive induction of VmlR by ribosome assembly defects. Rather, the specificity of induction is determined by the antibiotic's ability to stall the ribosome on the regulatory open reading frame located within the vmlR leader. Finally, we demonstrate the involvement of (p)ppGpp-mediated signalling in antibiotic-induced VmlR expression.

Small RNA-mediated regulation of the tet(M) resistance gene expression in Enterococcus faecium

We investigated the role of a novel small RNA expressed in Enterococcus faecium (named Ern0030). We revealed that ern0030 was encoded within the 5′untranslated region of tet(M), a gene conferring tetracycline resistance through ribosomal protection. By RACE mapping, we accurately determined the boundaries of ern0030, which corresponded to Ptet. This upstream sequence of tet(M), Ptet, was previously described within transcriptional attenuation mechanism. Here, Northern blot analyses revealed three transcripts of different lengths (ca. 230, 150 and 100 nucleotides) expressed from Ptet. Phenotypically, the total deletion of ern0030 conferred a decrease in tetracycline MICs that was consistent with gene expression data showing no significant tet(M) induction under tetracycline SIC in ern0030-deleted mutant as opposed to a 10-fold increase of tet(M) expression in the wild-type strain. We investigated the transcriptional attenuation mechanism by toeprint assay. Whereas the expected tet(M) ribosome-binding site (RBS) was detected, the RBS of the putative leader peptide was not highlighted by toeprint assay, suggesting the transcriptional attenuation was unlikely. Here, we demonstrate that Ern0030 has a role in regulation of tet(M) expression and propose a novel model of tet(M) regulation alternative or complementary to transcriptional attenuation.

Structure-seq2 probing of RNA structure upon amino acid starvation reveals both known and novel RNA switches in Bacillus subtilis.

RNA structure influences numerous processes in all organisms. In bacteria, these processes include transcription termination and attenuation, small RNA and protein binding, translation initiation, and mRNA stability, and can be regulated via metabolite availability and other stresses. Here we use Structure-seq2 to probe the in vivo RNA structurome of Bacillus subtilis grown in the presence and absence of amino acids. Our results reveal that amino acid starvation results in lower overall dimethyl sulfate (DMS) reactivity of the transcriptome, indicating enhanced protection owing to protein binding or RNA structure. Starvation-induced changes in DMS reactivity correlated inversely with transcript abundance changes. This correlation was particularly pronounced in genes associated with the stringent response and CodY regulons, which are involved in adaptation to nutritional stress, suggesting that RNA structure contributes to transcript abundance change in regulons involved in amino acid metabolism. Structure-seq2 accurately reported on four known amino acid-responsive riboswitches: T-box, SAM, glycine, and lysine riboswitches. Additionally, we discovered a transcription attenuation mechanism that reduces yfmG expression when amino acids are added to the growth medium. We also found that translation of a leader peptide (YfmH) encoded just upstream of yfmG regulates yfmG expression. Our results are consistent with a model in which a slow rate of yfmH translation caused by limitation of the amino acids encoded in YfmH prevents transcription termination in the yfmG leader region by favoring formation of an overlapping antiterminator structure. This novel RNA switch offers a way to simultaneously monitor the levels of multiple amino acids.

HflXr, a homolog of a ribosome-splitting factor, mediates antibiotic resistance

To overcome the action of antibiotics, bacteria have evolved a variety of different strategies, such as drug modification, target mutation, and efflux pumps. Recently, we performed a genome-wide analysis of Listeria monocytogenes gene expression after growth in the presence of antibiotics, identifying genes that are up-regulated upon antibiotic treatment. One of them, lmo0762, is a homolog of hflX, which encodes a heat shock protein that rescues stalled ribosomes by separating their two subunits. To our knowledge, ribosome splitting has never been described as an antibiotic resistance mechanism. We thus investigated the role of lmo0762 in antibiotic resistance. First, we demonstrated that lmo0762 is an antibiotic resistance gene that confers protection against lincomycin and erythromycin, and that we renamed hflXr (hflX resistance). We show that hflXr expression is regulated by a transcription attenuation mechanism relying on the presence of alternative RNA structures and a small ORF encoding a 14 amino acid peptide containing the RLR motif, characteristic of macrolide resistance genes. We also provide evidence that HflXr is involved in ribosome recycling in presence of antibiotics. Interestingly, L. monocytogenes possesses another copy of hflX, lmo1296, that is not involved in antibiotic resistance. Phylogenetic analysis shows several events of hflXr duplication in prokaryotes and widespread presence of hflXr in Firmicutes. Overall, this study reveals the Listeria hflXr as the founding member of a family of antibiotic resistance genes. The resistance conferred by this gene is probably of importance in the environment and within microbial communities.

Capture and Release of tRNA by the T-Loop Receptor in the Function of the T-Box Riboswitch.

In Gram-positive bacteria, the tRNA-dependent T-box riboswitch system regulates expression of amino acid biosynthetic and aminoacyl-tRNA synthetase genes through a transcription attenuation mechanism. Binding of uncharged tRNA "closes" the switch, allowing transcription read-through. Structural studies of the 100-nucleotide stem I domain reveal tRNA utilizes base pairing and stacking interactions to bind the stem, but little is known structurally about the 180-nucleotide riboswitch core (stem I, stem III, and antiterminator stem) in complex with tRNA or the mechanism of coupling of the intermolecular binding domains crucial to T-box function. Here we utilize solution structural and biophysical methods to characterize the interplay of the different riboswitch-tRNA contact points using Bacillus subtilis and Oceanobacillus iheyensis glycyl T-box and T-box:tRNA constructs. The data reveal that tRNA:riboswitch core binding at equilibrium involves only Specifier-anticodon and antiterminator-acceptor stem pairing. The elbow:platform stacking interaction observed in studies of the T-box stem I domain is released after pairing between the acceptor stem and the bulge in the antiterminator helix. The results are consistent with the model of T-box riboswitch:tRNA function in which tRNA is captured by stem I of the nascent mRNA followed by stabilization of the antiterminator helix and the paused transcription complex.

Modular Organization of the NusA- and NusG-Stimulated RNA Polymerase Pause Signal That Participates in the Bacillus subtilis trp Operon Attenuation Mechanism.

The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to the nascent transcript and blocks the formation of an antiterminator structure such that the formation of an overlapping intrinsic terminator causes termination in the 5' untranslated region (5' UTR). In the absence of bound TRAP, the antiterminator forms and transcription continues into the trp genes. RNA polymerase pauses at positions U107 and U144 in the 5' UTR. The general transcription elongation factors NusA and NusG stimulate pausing at both positions. NusG-stimulated pausing at U144 requires sequence-specific contacts with a T tract in the nontemplate DNA (ntDNA) strand within the paused transcription bubble. Pausing at U144 participates in a trpE translation repression mechanism. Since U107 just precedes the critical overlap between the antiterminator and terminator structures, pausing at this position is thought to participate in attenuation. Here we carried out in vitro pausing and termination experiments to identify components of the U107 pause signal and to determine whether pausing affects the termination efficiency in the 5' UTR. We determined that the U107 and U144 pause signals are organized in a modular fashion containing distinct RNA hairpin, U-tract, and T-tract components. NusA-stimulated pausing was affected by hairpin strength and the U-tract sequence, whereas NusG-stimulated pausing was affected by hairpin strength and the T-tract sequence. We also determined that pausing at U107 results in increased TRAP-dependent termination in the 5' UTR, implying that NusA- and NusG-stimulated pausing participates in the trp operon attenuation mechanism by providing additional time for TRAP binding.IMPORTANCE The expression of several bacterial operons is controlled by regulated termination in the 5' untranslated region (5' UTR). Transcription attenuation is defined as situations in which the binding of a regulatory molecule promotes transcription termination in the 5' UTR, with the default being transcription readthrough into the downstream genes. RNA polymerase pausing is thought to participate in several attenuation mechanisms by synchronizing the position of RNA polymerase with RNA folding and/or regulatory factor binding, although this has only been shown in a few instances. We found that NusA- and NusG-stimulated pausing participates in the attenuation mechanism controlling the expression of the Bacillus subtilis trp operon by increasing the TRAP-dependent termination efficiency. The pause signal is organized in a modular fashion containing RNA hairpin, U-tract, and T-tract components.

VirF Relieves the Transcriptional Attenuation of the Virulence Gene icsA of Shigella flexneri Affecting the icsA mRNA-RnaG Complex Formation.

VirF is the master activator of virulence genes of Shigella and its expression is required for the invasion of the human intestinal mucosa by pathogenic bacteria. VirF was shown to directly activate the transcription of virB and icsA, which encode two essential proteins involved in the pathogenicity process, by binding their promoter regions. In this study, we demonstrate by band shift, enzymatic probing and cross-linking experiments that VirF, in addition to DNA, can also bind the icsA transcript and RnaG, an antisense non-coding small RNA that promotes the premature termination of icsA mRNA through a transcriptional attenuation mechanism. Furthermore, we show that VirF binds in vitro also other species of RNAs, although with lower specificity. The existence of VirF–RnaG and VirF-icsA mRNA complexes is confirmed in a pulldown assay carried out under experimental conditions that very close reproduce the in vivo conditions and that allows immobilized VirF to “fish” out RnaG and icsA mRNA from a total RNA extract. The VirF binding sites identified on both icsA mRNA and RnaG contain a 13 nucleotides stretch (5′-UUUUaGYcUuUau-3′) that is the RNA-converted consensus sequence previously proposed for the VirF–DNA interaction. Band-shift assays with a synthetic RNA molecule whose sequence perfectly matches the consensus indicate that this signature plays a key role also in the VirF–RNA interaction, in particular when exposed in a stem–loop structure. To further explore the icsA-RnaG-VirF regulatory system, we developed an in vitro test (RNA–RNA Pairing Assay) in which pairing between icsA mRNA and synthetic RNAs that reproduce the individual stem–loop motifs of RnaG, was analyzed in the presence of VirF. This assay shows that this protein can prevent the formation of the kissing complex, defined as the initial nucleation points for RNA heteroduplex formation, between RnaG and icsA mRNA. Consistently, VirF alleviates the RnaG-mediated repression of icsA transcription in vitro. Therefore VirF, by hindering the icsA transcript-RnaG interaction, exhibits an activity opposed to that usually displayed by proteins, which generally assist the RNA–RNA interaction; this quite uncommon and new function and the regulatory implications of VirF as a potential RNA-binding protein are discussed.

NusG Is a Sequence-specific RNA Polymerase Pause Factor That Binds to the Non-template DNA within the Paused Transcription Bubble

NusG, referred to as Spt5 in archaeal and eukaryotic organisms, is the only transcription factor conserved in all three domains of life. This general transcription elongation factor binds to RNA polymerase (RNAP) soon after transcription initiation and dissociation of the RNA polymerase σ factor. Escherichia coli NusG increases transcription processivity by suppressing RNAP pausing, whereas Bacillus subtilis NusG dramatically stimulates pausing at two sites in the untranslated leader of the trpEDCFBA operon. These two regulatory pause sites participate in transcription attenuation and translational control mechanisms, respectively. Here we report that B. subtilis NusG makes sequence-specific contacts with a T-rich sequence in the non-template DNA (ntDNA) strand within the paused transcription bubble. NusG protects T residues of the recognition sequence from permanganate oxidation, and these T residues increase the affinity of NusG to the elongation complex. Binding of NusG to RNAP does not require interaction with RNA. These results indicate that bound NusG prevents forward movement of RNA polymerase by simultaneously contacting RNAP and the ntDNA strand. Mutational studies indicate that amino acid residues of two short regions within the NusG N-terminal domain are primarily responsible for recognition of the trp operon pause signals. Structural modeling indicates that these two regions are adjacent to each another in the protein. We propose that recognition of specific sequences in the ntDNA and stimulation of RNAP pausing is a conserved function of NusG-like transcription factors.

NusA-dependent transcription termination prevents misregulation of global gene expression.

Intrinsic transcription terminators consist of an RNA hairpin followed by a U-rich tract, and these signals can trigger termination without the involvement of additional factors. Although NusA is known to stimulate intrinsic termination in vitro, the in vivo targets and global impact of NusA are not known because it is essential for viability. Using genome-wide 3' end-mapping on an engineered Bacillus subtilis NusA depletion strain, we show that weak suboptimal terminators are the principle NusA substrates. Moreover, a subclass of weak non-canonical terminators was identified that completely depend on NusA for effective termination. NusA-dependent terminators tend to have weak hairpins and/or distal U-tract interruptions, supporting a model in which NusA is directly involved in the termination mechanism. Depletion of NusA altered global gene expression directly and indirectly via readthrough of suboptimal terminators. Readthrough of NusA-dependent terminators caused misregulation of genes involved in essential cellular functions, especially DNA replication and metabolism. We further show that nusA is autoregulated by a transcription attenuation mechanism that does not rely on antiterminator structures. Instead, NusA-stimulated termination in its 5' UTR dictates the extent of transcription into the operon, thereby ensuring tight control of cellular NusA levels.

SuhB is a novel ribosome associated protein that regulates expression of MexXY by modulating ribosome stalling in Pseudomonas aeruginosa.

Translation elongation is modulated by various ribosome-binding proteins. Environmental stresses, such as starvation and antibiotics, can cause stalling of bacterial ribosomes, which may alter gene expression through a transcription or translation attenuation mechanism. In Pseudomonas aeruginosa, the expression of MexXY multidrug efflux system, which plays a significant role in resistance against aminoglycoside antibiotics, is controlled by a translation surveillance mechanism. Stalling of ribosome at the PA5471 leader peptide (PA5471.1) mRNA leads to transcription of PA5471, which subsequently up-regulates the expression of MexXY. In this study, we found that mutation in a suhB gene leads to decreased susceptibility to aminoglycosides. Transcriptomic analysis revealed an up-regulation of MexXY and PA5471, which were demonstrated to be responsible for the decreased susceptibility of the suhB mutant. We further demonstrated that PA5471.1 is essential for the up-regulation of PA5471 in the suhB mutant. Co-immunoprecipitation assay revealed an interaction between SuhB and ribosome, suggesting a role of SuhB in translation. Indeed, higher amount of PA5471.1 mRNA was found to associate with ribosome isolated from the suhB mutant, indicating increased ribosome stalling. Therefore, this study identified SuhB as a novel ribosome associated protein that is involved in modulating ribosome activity.

Ribosomal protein L10(L12)4 autoregulates expression of the Bacillus subtilis rplJL operon by a transcription attenuation mechanism.

Ribosomal protein genes are often controlled by autoregulatory mechanisms in which a protein encoded in the operon can either bind to newly synthesized rRNA during rapid growth or to a similar target in its mRNA during poor growth conditions. The rplJL operon encodes the ribosomal L10(L12)4 complex. In Escherichia coli L10(L12)4 represses its translation by binding to the rplJL leader transcript. We identified three RNA structures in the Bacillus subtilis rplJL leader transcript that function as an anti-antiterminator, antiterminator or intrinsic terminator. Expression studies with transcriptional and translational fusions indicated that L10(L12)4 represses rplJL expression at the transcriptional level. RNA binding studies demonstrated that L10(L12)4 stabilizes the anti-antiterminator structure, while in vitro transcription results indicated that L10(L12)4 promotes termination. Disruption of anti-antiterminator, antiterminator or terminator function by competitor oligonucleotides in vitro and by mutations in vivo demonstrated that each structure functions as predicted. Thus, rplJL expression is regulated by an autogenous transcription attenuation mechanism in which L10(L12)4 binding to the anti-antiterminator structure promotes termination. We also found that translation of a leader peptide increases rplJL expression, presumably by inhibiting Rho-dependent termination. Thus, the rplJL operon of B. subtilis is regulated by transcription attenuation and antitermination mechanisms.

The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter.

Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan

A transcriptional attenuation mechanism regulates expression of the bacterial tnaCAB operon. This mechanism requires ribosomal arrest induced by the regulatory nascent TnaC peptide in response to free L-tryptophan (L-Trp). In this study we demonstrate, using genetic and biochemical analyses, that in Escherichia coli, TnaC residue I19 and 23S rRNA nucleotide A2058 are essential for the ribosome’s ability to sense free L-Trp. We show that the mutational change A2058U in 23S rRNA reduces the concentration dependence of L-Trp-mediated tna operon induction, whereas the TnaC I19L change suppresses this phenotype, restoring the sensitivity of the translating A2058U mutant ribosome to free L-Trp. These findings suggest that interactions between TnaC residue I19 and 23S rRNA nucleotide A2058 contribute to the creation of a regulatory L-Trp binding site within the translating ribosome.

Solution NMR determination of hydrogen bonding and base pairing between the glyQS T box riboswitch Specifier domain and the anticodon loop of tRNAGly

In Gram-positive bacteria the tRNA-dependent T box riboswitch regulates the expression of many amino acid biosynthetic and aminoacyl-tRNA synthetase genes through a transcription attenuation mechanism. The Specifier domain of the T box riboswitch contains the Specifier sequence that is complementary to the tRNA anticodon and is flanked by a highly conserved purine nucleotide that could result in a fourth base pair involving the invariant U33 of tRNA. We show that the interaction between the T box Specifier domain and tRNA consists of three Watson–Crick base pairs and that U33 confers stability to the complex through intramolecular hydrogen bonding. Enhanced packing within the Specifier domain loop E motif may stabilize the complex and contribute to cognate tRNA selection.

The tyrosyl-tRNA synthetase like gene located in the tyramine biosynthesis cluster of Enterococcus duransis transcriptionally regulated by tyrosine concentration and extracellular pH

BackgroundThe tyramine producer Enterococcus durans IPLA655 contains all the necessary genes for tyramine biosynthesis, grouped in the TDC cluster. This cluster includes tyrS, an aminoacyl-tRNA synthetase like gene.ResultsThis work shows that tyrS was maximally transcribed in absence of tyrosine at acidic pH, showing a greater than 10-fold induction in mRNA levels over levels occurring in presence of tyrosine. Mapping of the tyrS transcriptional start site revealed an unusually long untranslated leader region of 322 bp, which displays the typical features of the T box transcriptional attenuation mechanism. The tyrosine concentration regulation of tyrS was found to be mediated by a transcription antitermination system, whereas the specific induction at acidic pH was regulated at transcription initiation level.ConclusionsThe expression of the tyrS gene present in the TDC cluster of E. durans is transcriptionally regulated by tyrosine concentration and extracelular pH. The regulation is mediated by both an antitermination system and the promoter itself.

RNase Y is responsible for uncoupling the expression of translation factor IF3 from that of the ribosomal proteins L35 and L20 in Bacillus subtilis

RNase Y is a novel endoribonuclease affecting global mRNA metabolism. We show that this nuclease affects the expression of the Bacillus subtilis infC-rpmI-rplT operon, encoding translation initiation factor IF3 and the ribosomal proteins L35 and L20. This operon is autoregulated by a complex L20-dependent transcription attenuation mechanism. L20 binds to a phylogenetically conserved domain on the 5' untranslated region of the infC mRNA which mimics the L20 binding sites on 23S rRNA. We have identified a second promoter (P1) upstream of the previously identified promoter (P2). The P1, but not the P2, readthrough transcript is stabilized in a strain depleted for RNase Y. However, under these conditions infC biosynthesis is repressed threefold. We show that the unprocessed P1 transcript is non-functional for IF3 translation but fully competent to express the co-transcribed ribosomal protein genes. RNase Y cleavage of the P1 transcript creates an entry site for the 5'-3' exonucleolytic activity of RNase J1 which degrades the infC mRNA when translation initiation efficiency is low. A second RNase Y cleavage is crucial for initiating degradation of the prematurely terminated infC leader RNAs, including the L20 operator complex, which permits efficient recycling of the L20 protein.

Versatile RNA-sensing transcriptional regulators for engineering genetic networks

The widespread natural ability of RNA to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. Previous work in RNA synthetic biology has engineered RNA mechanisms that independently regulate multiple targets and integrate regulatory signals. However, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. In this work, we remove this requirement and expand the RNA synthetic biology toolkit by engineering three unique features of the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism. First, because the antisense RNA mechanism relies on RNA-RNA interactions, we show how the specificity of the natural system can be engineered to create variants that independently regulate multiple targets in the same cell. Second, because the pT181 mechanism controls transcription, we show how independently acting variants can be configured in tandem to integrate regulatory signals and perform genetic logic. Finally, because both the input and output of the attenuator is RNA, we show how these variants can be configured to directly propagate RNA regulatory signals by constructing an RNA-meditated transcriptional cascade. The combination of these three features within a single RNA-based regulatory mechanism has the potential to simplify the design and construction of genetic networks by directly propagating signals as RNA molecules.

Solution Structure of the K-Turn and Specifier Loop Domains from the Bacillus subtilis tyrS T-Box Leader RNA

In Gram-positive bacteria, the RNA transcripts of many amino acid biosynthetic and aminoacyl tRNA synthetase genes contain 5′ untranslated regions, or leader RNAs, that function as riboswitches. These T-box riboswitches bind cognate tRNA molecules and regulate gene expression by a transcription attenuation mechanism. The Specifier Loop domain of the leader RNA contains nucleotides that pair with nucleotides in the tRNA anticodon loop and is flanked on one side by a kink-turn (K-turn), or GA, sequence motif. We have determined the solution NMR structure of the K-turn sequence element within the context of the Specifier Loop domain. The K-turn sequence motif has several noncanonical base pairs typical of K-turn structures but adopts an extended conformation. The Specifier Loop domain contains a loop E structural motif, and the single-strand Specifier nucleotides stack with their Watson–Crick edges displaced toward the minor groove. Mg2+ leads to a significant bending of the helix axis at the base of the Specifier Loop domain, but does not alter the K-turn. Isothermal titration calorimetry indicates that the K-turn sequence causes a small enhancement of the interaction between the tRNA anticodon arm and the Specifier Loop domain. One possibility is that the K-turn structure is formed and stabilized when tRNA binds the T-box riboswitch and interacts with Stem I and the antiterminator helix. This motif in turn anchors the orientation of Stem I relative to the 3′ half of the leader RNA, further stabilizing the tRNA–T box complex.