Rho-independent Terminator Research Articles

Role of PatAB Transporter in Efflux of Levofloxacin in Streptococcus pneumoniae.

PatAB is an ABC bacterial transporter that facilitates the export of antibiotics and dyes. The overexpression of patAB genes conferring efflux-mediated fluoroquinolone resistance has been observed in several laboratory strains and clinical isolates of Streptococcus pneumoniae. Using transformation and whole-genome sequencing, we characterized the fluoroquinolone-resistance mechanism of one S. pneumoniae clinical isolate without mutations in the DNA topoisomerase genes. We identified the PatAB fluoroquinolone efflux-pump as the mechanism conferring a low-level resistance to ciprofloxacin (8 µg/mL) and levofloxacin (4 µg/mL). Genetic transformation experiments with different amplimers revealed that the entire patA plus the 5'-terminus of patB are required for levofloxacin-efflux. By contrast, only the upstream region of the patAB operon, plus the region coding the N-terminus of PatA containing the G39D, T43A, V48A and D100N amino acid changes, are sufficient to confer a ciprofloxacin-efflux phenotype, thus suggesting differences between fluoroquinolones in their binding and/or translocation pathways. In addition, we identified a novel single mutation responsible for the constitutive and ciprofloxacin-inducible upregulation of patAB. This mutation is predicted to destabilize the putative rho-independent transcriptional terminator located upstream of patA, increasing transcription of downstream genes. This is the first report demonstrating the role of the PatAB transporter in levofloxacin-efflux in a pneumoccocal clinical isolate.

Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities

Gene regulation by trans-acting small RNAs (sRNAs) has considerable advantages over other gene regulation strategies. However, synthetic sRNAs mainly take natural sRNAs (MicC or SgrS) as backbones and comprise three functional elements folding into two or more stem-loop structures: an mRNA base pairing region, an Hfq-binding structure, and a rho-independent terminator. Due to limited numbers of natural sRNAs and complicated backbone structures, synthetic sRNAs suffer from low activity programmability and poor structural modularity. Moreover, natural sRNA backbone sequences may increase the possibility of unwanted recombination. Here, we present a bottom-up approach for creating structure defined single-stem loop small non-coding RNAs (ssl-sRNAs), which contain a standardized scaffold of a 7 bp-stem-4 nt-loop-polyU-tail and a 24 nt basing pairing region covering the first eight codons. Particularly, ssl-sRNA requires no independent Hfq-binding structure, as the polyU tail fulfills the roles of binding Hfq. A thermodynamic-based scoring model and a web server sslRNAD (http://www.kangzlab.cn/) were developed for automated design of ssl-sRNAs with well-defined structures and programmable activities. ssl-sRNAs displayed weak polar effects when regulating polycistronic mRNAs. The ssl-sRNA designed by sslRNAD showed regulatory activities in both Escherichia coli and Bacillus subtilis. A streamlined workflow was developed for the construction of customized ssl-sRNA and ssl-sRNA libraries. As examples, the E. coli cell morphology was easily modified and new target genes of ergothioneine biosynthesis were quickly identified with ssl-sRNAs. ssl-sRNA and its designer sslRNAD enable researchers to rapidly design sRNAs for knocking down target genes.

Processing of the alaW alaX operon encoding the Ala2 tRNAs in Escherichia coli requires both RNase E and RNase P.

The alaW alaX operon encodes the Ala2 tRNAs, one of the two alanine tRNA isotypes in Escherichia coli. Our previous RNA-seq study showed that alaW alaX dicistronic RNA levels increased significantly in the absence of both RNase P and poly(A) polymerase I (PAP I), suggesting a role of polyadenylation in its stability. In this report, we show that RNase E initiates the processing of the primary alaW alaX precursor RNA by removing the Rho-independent transcription terminator, which appears to be the rate limiting step in the separation and maturation of the Ala2 pre-tRNAs by RNase P. Failure to separate the alaW and alaX pre-tRNAs by RNase P leads to poly(A)-mediated degradation of the dicistronic RNAs by polynucleotide phosphorylase (PNPase) and RNase R. Surprisingly, the thermosensitive RNase E encoded by the rne-1 allele is highly efficient in removing the terminator (>99%) at the nonpermissive temperature suggesting a significant caveat in experiments using this allele. Together, our data present a comprehensive picture of the Ala2 tRNA processing pathway and demonstrate that unprocessed RNase P substrates are degraded via a poly(A) mediated decay pathway.

Identification of Attenuators of Transcriptional Termination: Implications for RNA Regulation in Escherichia coli.

The regulatory function of many bacterial small RNAs (sRNAs) requires the binding of the RNA chaperone Hfq to the 3' portion of the sRNA intrinsic terminator, and therefore sRNA signaling might be regulated by modulating its terminator. Here, using a multicopy screen developed with the terminator of sRNA SgrS, we identified an sRNA gene (cyaR) and three protein-coding genes (cspD, ygjH, and rof) that attenuate SgrS termination in Escherichia coli. Analyses of CyaR and YgjH, a putative tRNA binding protein, suggested that the CyaR activity was indirect and the effect of YgjH was moderate. Overproduction of the protein attenuators CspD and Rof resulted in more frequent readthrough at terminators of SgrS and two other sRNAs, and regulation by SgrS of target mRNAs was reduced. The effect of Rof, a known inhibitor of Rho, was mimicked by bicyclomycin or by a rho mutant, suggesting an unexpected role for Rho in sRNA termination. CspD, a member of the cold shock protein family, bound both terminated and readthrough transcripts, stabilizing them and attenuating termination. By RNA sequencing analysis of the CspD overexpression strain, we found global effects of CspD on gene expression across some termination sites. We further demonstrated effects of endogenous CspD under slow growth conditions where cspD is highly expressed. These findings provided evidence of changes in the efficiency of intrinsic termination, confirming this as an additional layer of the regulation of sRNA signaling. IMPORTANCE Growing evidence suggests that the modulation of intrinsic termination and readthrough of transcription is more widespread than previously appreciated. For small RNAs, proper termination plays a critical role in their regulatory function. Here, we present a multicopy screen approach to identify factors that attenuate small RNA termination and therefore abrogate signaling dependent on the small RNA. This study highlights a new aspect of regulation of small RNA signaling as well as the modulation of intrinsic termination.

Comprehensive transcription terminator atlas for Bacillus subtilis.

The transcriptome-wide contributions of Rho-dependent and intrinsic (Rho-independent) transcription termination mechanisms in bacteria are unclear. By sequencing released transcripts in a wild-type strain and strains containing deficiencies in NusA, NusG and/or Rho (10 strains), we produced an atlas of terminators for the model Gram-positive bacterium Bacillus subtilis. We found that NusA and NusG stimulate 77% and 19% of all intrinsic terminators, respectively, and that both proteins participate in Rho-dependent termination. We also show that Rho stimulates termination at 10% of the intrinsic terminators in vivo. We recapitulated Rho-stimulated intrinsic termination at 5 terminators in vitro and found that Rho requires the KOW domain of NusG to stimulate this process at one of these terminators. Computational analyses of our atlas using RNAstructure, MEME suite and DiffLogo, combined with in vitro transcription experiments, revealed that Rho stimulates intrinsic terminators with weak hairpins and/or U-rich tracts by remodelling the RNA upstream of the intrinsic terminator to prevent the formation of RNA structures that could otherwise compete with the terminator hairpin. We also identified 56 putative examples of 'hybrid Rho-dependent termination', wherein classical Rho-dependent termination occurs after readthrough of a Rho-stimulated intrinsic terminator.

Structural insights into RNA-mediated transcription regulation in bacteria

RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination.

Intrinsic and Rho-dependent termination cooperate for efficient transcription termination at 3’ untranslated regions

The intrinsic, and the Rho-dependent mechanisms of transcription termination are conserved in bacteria. Generally, the two mechanisms have been illustrated as two independent pathways occurring in the 3′ ends of different genes with contrasting requirements to halt RNA synthesis. However, a majority of intrinsic terminators terminate transcription inefficiently leading to transcriptional read-through. The unwanted transcription in the downstream region beyond the terminator would have undesired consequences. To prevent such transcriptional read-through, bacteria must have evolved ways to terminate transcription more efficiently at or near the termination sites. We describe the participation of both the mechanisms, where intrinsic terminator and Rho factor contribute to prevent transcriptional read-through. Contribution from both the termination processes is demonstrated at the downstream regions of the genes both in vitro and in vivo in mycobacteria. Distinct patterns of cooperation between the two modes of termination were observed at the 3’ untranslated regions of the genes to ensure efficient termination. We demonstrate similar mode of operation between the two termination processes in Escherichia coli suggesting a likely prevalence of this cooperation across bacteria. The reporter system developed to assess the Rho – intrinsic termination collaboration in vivo for mycobacteria and E. coli can readily be applied to other bacteria.

Incoherent dual regulation by a SAM-II riboswitch controlling translation at a distance

ABSTRACT In Sinorhizobium meliloti, the methionine biosynthesis genes metA and metZ are preceded by S-adenosyl-L-methionine (SAM) riboswitches of the SAM-II class. Upon SAM binding, structural changes in the metZ riboswitch were predicted to cause transcriptional termination, generating the sRNA RZ. By contrast, the metA riboswitch was predicted to regulate translation from an AUG1 codon. However, downstream of the metA riboswitch, we found a putative Rho-independent terminator and an in-frame AUG2 codon, which may contribute to metA regulation. We validated the terminator between AUG1 and AUG2, which generates the sRNA RA1 that is processed to RA2. Under high SAM conditions, the activities of the metA and metZ promoters and the steady-state levels of the read-through metA and metZ mRNAs were decreased, while the levels of the RZ and RA2 sRNAs were increased. Under these conditions, the sRNAs and the mRNAs were stabilized. Reporter fusion experiments revealed that the Shine–Dalgarno (SD) sequence in the metA riboswitch is required for translation, which, however, starts 74 nucleotides downstream at AUG2, suggesting a novel translation initiation mechanism. Further, the reporter fusion data supported the following model of RNA-based regulation: Upon SAM binding by the riboswitch, the SD sequence is sequestered to downregulate metA translation, while the mRNA is stabilized. Thus, the SAM-II riboswitches fulfil incoherent, dual regulation, which probably serves to ensure basal metA and metZ mRNA levels under high SAM conditions. This probably helps to adapt to changing conditions and maintain SAM homoeostasis.

Structural basis of AlpA-dependent transcription antitermination.

AlpA positively regulates a programmed cell death pathway linked to the virulence of Pseudomonas aeruginosa by recognizing an AlpA binding element within the promoter, then binding RNA polymerase directly and allowing it to bypass an intrinsic terminator positioned downstream. Here, we report the single-particle cryo-electron microscopy structures of both an AlpA-loading complex and an AlpA-loaded complex. These structures indicate that the C-terminal helix-turn-helix motif of AlpA binds to the AlpA binding element and that the N-terminal segment of AlpA forms a narrow ring inside the RNA exit channel. AlpA was also revealed to render RNAP resistant to termination signals by prohibiting RNA hairpin formation in the RNA exit channel. Structural analysis predicted that AlpA, 21Q, λQ and 82Q share the same mechanism of transcription antitermination.

Inverse folding based pre-training for the reliable identification of intrinsic transcription terminators.

It is well-established that neural networks can predict or identify structural motifs of non-coding RNAs (ncRNAs). Yet, the neural network based identification of RNA structural motifs is limited by the availability of training data that are often insufficient for learning features of specific ncRNA families or structural motifs. Aiming to reliably identify intrinsic transcription terminators in bacteria, we introduce a novel pre-training approach that uses inverse folding to generate training data for predicting or identifying a specific family or structural motif of ncRNA. We assess the ability of neural networks to identify secondary structure by systematic in silico mutagenesis experiments. In a study to identify intrinsic transcription terminators as functionally well-understood RNA structural motifs, our inverse folding based pre-training approach significantly boosts the performance of neural network topologies, which outperform previous approaches to identify intrinsic transcription terminators. Inverse-folding based pre-training provides a simple, yet highly effective way to integrate the well-established thermodynamic energy model into deep neural networks for identifying ncRNA families or motifs. The pre-training technique is broadly applicable to a range of network topologies as well as different types of ncRNA families and motifs.

Development and Application of Transcription Terminators for Polyhydroxylkanoates Production in Halophilic Halomonas bluephagenesis TD01.

Halomonas bluephagenesis TD01 is one of the ideal chassis for low-cost industrial production based on “Next Generation Industrial Biotechnology,” yet the limited genetically regulatory parts such as transcriptional terminators, which are crucial for tuned regulations on gene expression, have hampered the engineering and applications of the strain. In this study, a series of intrinsic Rho-independent terminators were developed by either genome mining or rational design, and seven of them proved to exhibit higher efficiencies than the canonical strong T7 terminator, among which three terminators displayed high efficiencies over 90%. A preliminary modeling on the sequence-efficiency relationship of the terminators suggested that the poly U sequence regularity, the length and GC content of the stem, and the number and the size of hairpin loops remarkably affected the termination efficiency (TE). The rational and de novo designs of novel synthetic terminators based on the sequence-efficiency relationship and the “main contributor” engineering strategy proved to be effective, and fine-tuned polyhydroxylkanoates production was also achieved by the regulation of these native or synthetic terminators with different efficiencies. Furthermore, a perfectly positive correlation between the promoter activity and the TE was revealed in our study. The study enriches our knowledge of transcriptional termination via its sequence–strength relationship and enables the precise regulation of gene expression and PHA synthesis by intrinsic terminators, contributing to the extensive applications of H. bluephagenesis TD01 in the low-cost production of various chemicals.

Sequence and thermodynamic characteristics of terminators revealed by FlowSeq and the discrimination of terminators strength

The intrinsic terminator in prokaryotic forms secondary RNA structure and terminates the transcription. However, leaking transcription is common due to varied terminator strength. Besides of the representative hairpin and U-tract structure, detailed sequence and thermodynamic features of terminators were not completely clear, and the effect of terminator on the upstream gene expression was unclearly. Thus, it is still challenging to use terminator to control expression with higher precision. Here, in E. Coli, we firstly determined the effect of the 3′-end sequences including spacer sequences and terminator sequences on the expression of upstream and downstream genes. Secondly, terminator mutation library was constructed, and the thermodynamic and sequence features differing in the termination efficiency were analyzed using the FlowSeq technique. The result showed that under the regulation of terminators, a negative correlation was presented between the expression of upstream and downstream genes (r=−0.60), and the terminators with lower free energy corelated with higher upstream gene expression. Meanwhile, the terminator with longer stem length, more compact loop and perfect U-tract structure was benefit to the transcription termination. Finally, a terminator strength classification model was established, and the verification experiment based on 20 synthetic terminators indicated that the model can distinguish strong and weak terminators to certain extent. The results help to elucidate the role of terminators in gene expression, and the key factors identified are crucial for rational design of terminators, and the model provided a method for terminator strength prediction.

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.

Characterization of Five Purine Riboswitches in Cellular and Cell-Free Expression Systems.

Bacillus subtilis employs five purine riboswitches for the control of purine de novo synthesis and transport at the transcription level. All of them are formed by a structurally conserved aptamer, and a variable expression platform harboring a rho-independent transcription terminator. In this study, we characterized all five purine riboswitches under the context of active gene expression processes both in vitro and in vivo. We identified transcription pause sites located in the expression platform upstream of the terminator of each riboswitch. Moreover, we defined a correlation between in vitro transcription readthrough and in vivo gene expression. Our in vitro assay demonstrated that the riboswitches operate in the micromolar range of concentration for the cognate metabolite. Our in vivo assay showed the dynamics of the control of gene expression by each riboswitch. This study deepens the knowledge of the regulatory mechanism of purine riboswitches.

The RNA-bound proteome of MRSA reveals post-transcriptional roles for helix-turn-helix DNA-binding and Rossmann-fold proteins

RNA-binding proteins play key roles in controlling gene expression in many organisms, but relatively few have been identified and characterised in detail in Gram-positive bacteria. Here, we globally analyse RNA-binding proteins in methicillin-resistant Staphylococcus aureus (MRSA) using two complementary biochemical approaches. We identify hundreds of putative RNA-binding proteins, many containing unconventional RNA-binding domains such as Rossmann-fold domains. Remarkably, more than half of the proteins containing helix-turn-helix (HTH) domains, which are frequently found in prokaryotic transcription factors, bind RNA in vivo. In particular, the CcpA transcription factor, a master regulator of carbon metabolism, uses its HTH domain to bind hundreds of RNAs near intrinsic transcription terminators in vivo. We propose that CcpA, besides acting as a transcription factor, post-transcriptionally regulates the stability of many RNAs.

Optimization of RNA Display Using GC‐Clamp Modifications to Improve Genetic Detection of RNA‐Protein Interactions

Non‐coding small regulatory RNAs (sRNAs) have an important role in bacterial stress responses. The binding of sRNAs to their target mRNAs is facilitated by RNA‐binding proteins such as Hfq or ProQ. The Berry Lab has developed a bacterial three‐hybrid (B3H) assay to detect the binding of RNA with both of these RNA chaperones in vivo, by connecting the strength of an RNA‐protein interaction to the expression of a reporter gene. The interaction between a “prey” protein fused to the α‐subunit of RNA polymerase (RNAP) and a “bait” RNA tethered upstream of a test promoter stabilizes the binding of RNAP and increases transcription of the reporter gene lacZ. Despite the promise of the B3H system and its success in detecting many high‐affinity interactions, low signal‐to‐noise for other RNA‐protein interactions currently limits the broader utility of the assay. Computational structure predictions suggested that certain RNAs of interest could misfold when hybridized with other components of the hybrid RNA construct, e.g. an MS2 hairpin (MS2hp) or an exogenous intrinsic terminator. Such misfolding would likely disrupt a bait RNA’s interaction with the prey protein. To avoid this limitation, this study aims to optimize the hybrid RNA construct to improve the breadth of detectable interactions in the B3H assay. To this end, we designed new hybrid RNA constructs with the addition of a GC‐clamp – a short insert of guanines (G) and cytosines (C) flanking a region of interest – to promote proper folding and optimal display of RNA. Several sRNAs and 5’UTRs were cloned into GC‐clamp designs and their in vivo interactions with Hfq were tested in the B3H assay. Preliminary results demonstrate the promise of a short GC‐clamp in improving the B3H signal for many sRNA‐Hfq interactions, and we are currently working to test these GC constructs with additional RNAs. Increasing the sensitivity and generalizability of the B3H assay to study bacterial RNA‐protein interactions will help shed light on the molecular mechanisms of RNA‐chaperone proteins and the important processes in bacteria they regulate, such as adaptation to stress, biofilm formation and virulence.

Synthetic 3'-UTR valves for optimal metabolic flux control in Escherichia coli.

As the design of genetic circuitry for synthetic biology becomes more sophisticated, diverse regulatory bioparts are required. Despite their importance, well-characterized 3′-untranslated region (3′-UTR) bioparts are limited. Thus, transcript 3′-ends require further investigation to understand the underlying regulatory role and applications of the 3′-UTR. Here, we revisited the use of Term-Seq in the Escherichia coli strain K-12 MG1655 to enhance our understanding of 3′-UTR regulatory functions and to provide a diverse collection of tunable 3′-UTR bioparts with a wide termination strength range. Comprehensive analysis of 1,629 transcript 3′-end positions revealed multiple 3′-termini classes generated through transcription termination and RNA processing. The examination of individual Rho-independent terminators revealed a reduction in downstream gene expression over a wide range, which led to the design of novel synthetic metabolic valves that control metabolic fluxes in branched pathways. These synthetic metabolic valves determine the optimal balance of heterologous pathways for maximum target biochemical productivity. The regulatory strategy using 3′-UTR bioparts is advantageous over promoter- or 5′-UTR-based transcriptional control as it modulates gene expression at transcription levels without trans-acting element requirements (e.g. transcription factors). Our results provide a foundational platform for 3′-UTR engineering in synthetic biology applications.

Complete Genome Sequence of Enterococcus faecalis Siphophage Sigurd.

ABSTRACTEnterococcus faecalis is associated with antibiotic-resistant infections, and this study presents E. faecalis siphophage Sigurd. The 41,811-bp Sigurd genome is divided into two arms defined by long convergent predicted transcription units that are separated by a bidirectional rho-independent terminator. Sigurd has a small terminase that is closely related to Bacillus subtilis cos phage phi105.

The Role and Targets of the RNA-Binding Protein ProQ in the Gram-Negative Bacterial Pathogen Pasteurella multocida.

The Gram-negative pathogen Pasteurella multocida is the causative agent of many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by an RNA-binding protein such as ProQ or Hfq. In Escherichia coli and a small number of other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to the 3' stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ in regulating P. multocida transcript abundance and identify the RNA targets to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-cross-linking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilizes, ProQ-dependent sRNAs and transfer RNAs in P. multocida via adenosine-enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterized, and these analyses showed that ProQ bound within the coding sequence of the transcript PmVP161_1121, encoding an uncharacterized protein, and within the 3' region of the putative sRNA Prrc13. IMPORTANCE Regulation in P. multocida involving the RNA-binding protein Hfq is required for hyaluronic acid capsule production and virulence. This study further expands our understanding of riboregulation by examining the role of a second RNA-binding protein, ProQ, in transcript regulation and abundance in P. multocida.

Internal Transcription Terminators Control Stoichiometry of ABC Transporters in Cellulolytic Clostridia.

ABSTRACTThe extracellular substrate-binding proteins (SBPs) of ATP-binding cassette (ABC) importers tend to be expressed in excess relative to their cognate translocators, but how the stoichiometry of ABC transporters is controlled remains unclear. Here, we elucidated a mechanism contributing to differential gene expression in operons encoding ABC importers by employing cellulolytic Clostridia species, specifically Ruminiclostridium cellulolyticum. We found that there were usually stem-loop structures downstream of SBP genes, which could prematurely terminate the transcription of ABC importers and were putative internal intrinsic terminators, resulting in high transcript levels of upstream SBP genes and low transcript levels of downstream cognate translocator genes. This was determined by their termination efficiencies. Internal terminators had a lower U content in their 3′ U-rich tracts and longer GC-rich stems, which distinguishes them from canonical terminators and potentially endows them with special termination efficiencies. The pairing of U-rich tracts and the formation of unpaired regions in these internal terminators contributed to their folding energies, affecting the stability of their upstream SBP transcripts. Our findings revealed a strategy of internal transcriptional terminators controlling in vivo stoichiometry of their flanking transcripts.IMPORTANCE Operons encoding protein complexes or metabolic pathways usually require fine-tuned gene expression ratios to create and maintain the appropriate stoichiometry for biological functions. In this study, a strategy for controlling differential expression of genes in an operon was proposed by utilizing ABC importers from Ruminiclostridium cellulolyticum. We found that a stem-loop structure is introduced into the intergenic regions of operons encoding ABC importers as the putative internal terminator, which results in the premature termination of transcription. Consequently, the stoichiometric ratio of genes flanking terminators is precisely determined by their termination efficiencies and folding energies at the transcriptional level. Thus, it can be utilized as a promising synthetic biology tool to control the differential expression of genes in an operon.