Terminator Stem Research Articles

Recognition of transcription terminators during retrotransposition: How to keep a group II intron quiet

Chung etal. recently presented the structure of a primitive group IIC intron with its DNA target, which reveals the structural requirements that this class of intron uses to recognize a transcription terminator stem loop at the DNA level for insertion during retrotransposition.

Competition between Ligand Binding and Transcription Rate Modulates Riboswitch-Mediated Regulation of Transcription

Riboswitches are genetic elements mostly found in the 5’ untranslated region of bacterial mRNAs, which act to control gene expression through small molecule-mediated conformational changes. In particular, the fluoride riboswitch, found in Bacillus cereus, regulates transcription upon binding of the small negatively charged ion, fluoride. Binding of the ligand to the riboswitch prevents the formation of a terminator hairpin, which in turn promotes active transcription of the downstream crcBgene. Previous studies have determined the structure of the riboswitch alone and in its transcriptional elongation complex (EC), but little is known about the impact of co-transcriptional folding of the riboswitch on termination of transcription. Using in vitro transcription assays, we found that fluoride and the transcription rate each modulate the termination efficiency in dependence of the riboswitch while competing with each other. Interestingly, we found that a downstream transcriptional pause of RNAP is modulated by the presence of fluoride, leading to efficient termination when the ligand is absent. Here, we used Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to probe the conformational dynamics of the terminator stem as a function of ligand binding and transcription rate. Interestingly, the terminator stem in a paused corresponding nascent run-off transcript is more stable, showing that the presence of RNAP negatively impacts this particular RNA structure at the pause site. Finally, we observed that a fast transcription rate negatively impacts the terminator stem formation showing the profound impact of the transcription process into riboswitch-mediated regulation of gene expression. These results will be presented in light of future works using transcription factors that affect the transcription rate. This work provides unprecedented insights into the powerful mechanism by which RNA structure can modulate the function of RNA polymerase and vice versa.

A bacterial riboswitch class for the thiamin precursor HMP-PP employs a terminator-embedded aptamer.

We recently implemented a bioinformatics pipeline that can uncover novel, but rare, riboswitch candidates as well as other noncoding RNA structures in bacteria. A prominent candidate revealed by our initial search efforts was called the 'thiS motif' because of its frequent association with a gene coding for the ThiS protein, which delivers sulfur to form the thiazole moiety of the thiamin precursor HET-P. In the current report, we describe biochemical and genetic data demonstrating that thiS motif RNAs function as sensors of the thiamin precursor HMP-PP, which is fused with HET-P ultimately to form the final active coenzyme thiamin pyrophosphate (TPP). HMP-PP riboswitches exhibit a distinctive architecture wherein an unusually small ligand-sensing aptamer is almost entirely embedded within an otherwise classic intrinsic transcription terminator stem. This arrangement yields remarkably compact genetic switches that bacteria use to tune the levels of thiamin precursors during the biosynthesis of this universally distributed coenzyme.

Sensitive and Specific Detection of Ligands Using Engineered Riboswitches

Riboswitches are RNA elements found in non‐coding regions of messenger RNAs that regulate gene expression through a ligand‐triggered conformational change (for review, see ref. 1). In, B. subtilis and other gram positive bacteria, purine metabolism is regulated by riboswitches that bind to guanine (2). The best studied of the guanine riboswitches regulates the transcription of the B. subtilis xpt‐pbuX operon. Guanine binding to this riboswitch inhibits transcription by stabilizing an alternative conformation that allows formation of a premature transcriptional terminator. In order to study guanine‐triggered structure switching, we have developed a fluorescence quenching assay for the function of the guanine riboswitch. Our system consists of the guanine‐binding domain (aptamer domain) of the riboswitch (xpt RNA), and two DNA oligonucleotides that are related to the 5′ and 3′ halves of the terminator stem (5′T and 3′T). xpt RNA is labeled at its 3′ end with a fluorophore and 5′T is labeled at its 5′ end with a fluorescence quencher. When 5′T is annealed to a complementary sequence at the 3′ end of xpt RNA, fluorescence is quenched. Guanine binding causes 5′T to dissociate from xpt RNA and anneal to 3′T. This strand‐exchange reaction causes an increase in fluorescence intensity as the quencher moves away from the fluorophore. Using this assay, we reproducibly detected as little as 5 nM guanine. We are currently using the same strategy to adapt a 2′‐deoxyguanosine riboswitch (3) for use as a probe that can detect its ligand in vitro. In addition, we have used our strand‐exchange reaction to develop a method for in vitro selection of variant riboswitches with altered ligand specificity. The results of our latest experiments will be discussed.Support or Funding InformationDefense Threat Reduction Agency (DTRA), Office of Naval Research (ONR), Chemistry Department, U.S, Naval Academy

Measurement and modeling of intrinsic transcription terminators

The reliable forward engineering of genetic systems remains limited by the ad hoc reuse of many types of basic genetic elements. Although a few intrinsic prokaryotic transcription terminators are used routinely, termination efficiencies have not been studied systematically. Here, we developed and validated a genetic architecture that enables reliable measurement of termination efficiencies. We then assembled a collection of 61 natural and synthetic terminators that collectively encode termination efficiencies across an ∼800-fold dynamic range within Escherichia coli. We simulated co-transcriptional RNA folding dynamics to identify competing secondary structures that might interfere with terminator folding kinetics or impact termination activity. We found that structures extending beyond the core terminator stem are likely to increase terminator activity. By excluding terminators encoding such context-confounding elements, we were able to develop a linear sequence-function model that can be used to estimate termination efficiencies (r = 0.9, n = 31) better than models trained on all terminators (r = 0.67, n = 54). The resulting systematically measured collection of terminators should improve the engineering of synthetic genetic systems and also advance quantitative modeling of transcription termination.

RNA evolution conjectured from tRNA and riboswitches

The structures of tRNAs and riboswitches comprise distinct domains. A tRNA molecule is L-shaped, and each arm may reflect its evolution. The amino acid-nonspecific minihelix domain may have emerged before the amino acid- specific anticodon-containing domain was acquired. The glycine riboswitch of Bacillus subtilis functions in a glycine- independent manner in the presence of polyethylene glycol or ethylene glycol. The effect is dependent only on the existence of a terminator stem within the expression platform of the riboswitch and is indepen- dent of the aptamer domain. Similar to the hypothesis that explains the evolution of tRNA function, the specificity of riboswitch functional domains may have increased during evolution. Collectively, these views provide new perspectives on the evolution of RNA.

Genetic Analysis of Riboswitch-mediated Transcriptional Regulation Responding to Mn2+ in Salmonella

Riboswitches are a class of cis-acting regulatory RNAs normally characterized from the 5'-UTR of bacterial transcripts that bind a specific ligand to regulate expression of associated genes by forming alternative conformations. Here, we present a riboswitch that contributes to transcriptional regulation through sensing Mn(2+) in Salmonella typhimurium. We characterized a 5'-UTR (UTR1) from the mntH locus encoding a Mn(2+) transporter, which forms a Rho-independent terminator to implement transcription termination with a high Mn(2+) selectivity both in vivo and in vitro. Nucleotide substitutions that cause disruption of the terminator interfere with the regulatory function of UTR1. RNA probing analyses outlined a specific UTR1 conformation that favors the terminator structure in Mn(2+)-replete condition. Switch sequence GCUAUG can alternatively base pair duplicated hexanucleotide CAUAGC to form either a pseudoknot or terminator stem. Mn(2+), but not Mg(2+), and Ca(2+), can enhance cleavage at specific nucleotides in UTR1. We conclude that UTR1 is a riboswitch that senses cytoplasmic Mn(2+) and therefore participates in Mn(2+)-responsive mntH regulation in Salmonella. This riboswitch domain is also conserved in several Gram-negative enteric bacteria, indicating that this Mn(2+)-responsive mechanism could have broader implications in bacterial gene expression. Additionally, a high level of cytoplasmic Mn(2+) can down-regulate transcription of the Salmonella Mg(2+) transporter mgtA locus in a Mg(2+) riboswitch-dependent manner. On the other hand, these two types of cation riboswitches do not share similarity at the primary or secondary structural levels. Taken together, characterization of Mn(2+)-responsive riboswitches should expand the scope of RNA regulatory elements in response to inorganic ions.

Time-Resolved and Dynamic Studies of Riboswitches

Riboswitches are a class of regulatory RNA molecules widely distributed mostly across prokaryotic organisms. The discovery of the first riboswitches, by the Breaker lab and others, opened the door to a previously unimagined layer of genetic regulation. The majority of riboswitches undergo large scale conformational rearrangements upon binding target metabolites in their aptamer domain. Riboswitches are found predominantly in the 5' untranslated region of a number of genes. As a nascent strand is being transcribed by an RNA polymerase, the switch can fold to either occlude the expression platform or to expose it. The former leads to termination of the transcript, while the latter leads to gene expression. It is hypothesized that the concentration of the riboswitch's target metabolite governs gene expression. Thus, if the switch has bound its target metabolite prior to moving past the critical terminator stem, gene expression is regulated. Conversely, if the polymerase moves past the terminator stem before binding the target metabolite, the riboswitch and metabolite will not have time to reach equilibrium before an on/off decision is made. Accordingly, the mechanism of regulation may be highly dependent on the kinetic on-rate. For these reasons, understanding the dynamics of the riboswitch both on a global and local level is critical to determining the molecular mechanism by which riboswitches regulate gene expression. NMR T1, T2, NOE, cross-correlation, and dispersion experiments would be presented to delineate the site-specific dynamics involved in riboswitch function. These local dynamics measurements are combined with other biophysical techniques that we use to track global structural changes on a wide range of time-scales. These studies would likely provide a glimpse of the conformational switching necessary for riboswitch function.

Glycols modulate terminator stem stability and ligand-dependency of a glycine riboswitch

The Bacillus subtilis glycine riboswitch comprises tandem glycine-binding aptamers and a putative terminator stem followed by the gcvT operon. Gene expression is regulated via the sensing of glycine. However, we found that the riboswitch behaves in a “glycine-independent” manner in the presence of polyethylene glycol (PEG) and ethylene glycol. The effect is related to the formation of a terminator stem within the expression platform under such conditions. The results revealed that increasing PEG stabilized the structure of the terminator stem. By contrast, the addition of ethylene glycol destabilized the terminator stem. PEG and ethylene glycol have opposite effects on transcription as well as on stable terminator stem formation. The glycine-independency of the riboswitch and the effects of such glycols might shed light on the evolution of riboswitches.

Measurement and modeling of intrinsic transcription terminators

The reliable forward engineering of genetic systems remains limited by the ad hoc reuse of many types of basic genetic elements. Although a few intrinsic prokaryotic transcription terminators are used routinely, termination efficiencies have not been studied systematically. Here, we developed and validated a genetic architecture that enables reliable measurement of termination efficiencies. We then assembled a collection of 61 natural and synthetic terminators that collectively encode termination efficiencies across an ∼800-fold dynamic range within Escherichia coli. We simulated co-transcriptional RNA folding dynamics to identify competing secondary structures that might interfere with terminator folding kinetics or impact termination activity. We found that structures extending beyond the core terminator stem are likely to increase terminator activity. By excluding terminators encoding such context-confounding elements, we were able to develop a linear sequence-function model that can be used to estimate termination efficiencies (r = 0.9, n = 31) better than models trained on all terminators (r = 0.67, n = 54). The resulting systematically measured collection of terminators should improve the engineering of synthetic genetic systems and also advance quantitative modeling of transcription termination.

Revelation of Anti-Termination Mechanism - Through Structural and Functional Analyses of HutP

Regulating gene expression directly at the mRNA level represents a novel approach in the control of cellular processes in all organisms. In this respect, RNA-binding proteins, while in the presence of their cognate ligands, play a key role by targeting the mRNA to regulate its expression through attenuation or anti-termination mechanisms. Although many proteins are known to use these mechanisms in the regulation of gene expression, no structural insights have been revealed, to date, to explain how these proteins trigger the conformation for the recognition of RNA. This review de- scribes the HutP mediated anti-termination mechanism by combining the in vivo, in vitro and X-ray analyses of the acti- vated conformation of HutP, initiated by the coordination of L-histidine and Mg 2+ ions, based on our previous and re- cently solved crystal structures (uncomplexed HutP, HutP-Mg 2+ , HutP-L-histidine, HutP-Mg 2+ -L-histidine, HutP-Mg 2+ -L- histidine-RNA (21-mer and 55-mer)). In this anti-termination process, HutP initiates destabilization at the 5'-end of its mRNA by binding to the first UAG-rich region and then accesses the second UAG-rich region, located downstream of the stable G-C-rich segment of the terminator stem. By this mode of action, HutP appears to disrupt the G-C rich terminator stem loop, and allow the RNA polymerase to pass through the destabilized terminator, thus it prevents premature termina- tion of transcription in the RNA segment preceding the regions encoding for the genes responsible for histidine degrada- tion.

A variant riboswitch aptamer class for S-adenosylmethionine common in marine bacteria

Riboswitches that sense S-adenosylmethionine (SAM) are widely distributed throughout a variety of bacterial lineages. Four classes of SAM-binding riboswitches have been reported to date, constituting the most diverse collection of riboswitch classes that sense the same compound. Three of these classes, termed SAM-I, SAM-II, and SAM-III represent unique structures that form distinct binding pockets for the ligand. SAM-IV riboswitches carry different conserved sequence and structural features compared to other SAM riboswitches, but nucleotides and substructures corresponding to the ligand binding pocket are identical to SAM-I aptamers. In this article, we describe a fifth class of SAM binding aptamer, which we have termed SAM-V. SAM-V was discovered by analyzing GC-rich intergenic regions preceding metabolic genes in the marine alpha-proteobacterium "Candidatus Pelagibacter ubique." Although the motif is nearly unrepresented in cultured bacteria whose genomes have been completely sequenced, SAM-V is prevalent in marine metagenomic sequences. The consensus sequence and structure of SAM-V show some similarities to that of the SAM-II riboswitch, and it is likely that the two aptamers form similar ligand binding pockets. In addition, we identified numerous examples of a tandem SAM-II/SAM-V aptamer architecture. In this arrangement, the SAM-II aptamer is always positioned 5' of the SAM-V aptamer and the SAM-II aptamer is followed by a predicted intrinsic transcription terminator stem. The SAM-V aptamer, however, appears to use a ribosome binding site occlusion mechanism for genetic regulation. This tandem riboswitch arrangement exhibits an architecture that can potentially control both the transcriptional and translational stages of gene expression.

Insights into anti-termination regulation of the hut operon in Bacillus subtilis: importance of the dual RNA-binding surfaces of HutP.

The anti-termination protein, HutP, regulates the gene expression of the hut (histidine utilization) operon of Bacillus subtilis, by destabilizing the hut terminator RNA located upstream of the coding region encoding l-histidine degradation enzymes. On the basis of biochemical, in vivo and X-ray structural analyses, we now report that HutP uses its dual RNA-binding surfaces to access two XAG-rich regions (sites I and II) within the terminator RNA to mediate the destabilization process. In this process, HutP initiates destabilization at the 5′-end of its mRNA by binding to the first XAG-rich region (site I) and then accesses the second XAG-rich region (site II), located downstream of the stable G-C-rich segment of the terminator stem. By this action, HutP appears to disrupt the G-C-rich terminator stem, and thus prevents premature termination of transcription in the RNA segment preceding the regions encoding for the histidine degradation enzymes.

In vitro analysis of the interaction between the small RNA SR1 and its primary target ahrC mRNA

Small regulatory RNAs (sRNAs) from bacterial chromosomes became the focus of research over the past five years. However, relatively little is known in terms of structural requirements, kinetics of interaction with their targets and degradation in contrast to well-studied plasmid-encoded antisense RNAs. Here, we present a detailed in vitro analysis of SR1, a sRNA of Bacillus subtilis that is involved in regulation of arginine catabolism by basepairing with its target, ahrC mRNA. The secondary structures of SR1 species of different lengths and of the SR1/ahrC RNA complex were determined and functional segments required for complex formation narrowed down. The initial contact between SR1 and its target was shown to involve the 5′ part of the SR1 terminator stem and a region 100 bp downstream from the ahrC transcriptional start site. Toeprinting studies and secondary structure probing of the ahrC/SR1 complex indicated that SR1 inhibits translation initiation by inducing structural changes downstream from the ahrC RBS. Furthermore, it was demonstrated that Hfq, which binds both SR1 and ahrC RNA was not required to promote ahrC/SR1 complex formation but to enable the translation of ahrC mRNA. The intracellular concentrations of SR1 were calculated under different growth conditions.

Folding of the Adenine Riboswitch

The pbuE adenine riboswitch undergoes metal ion-dependent folding that involves a loop-loop interaction. Binding of 2-aminopurine to the aptamer domain strongly correlates with the ability of the loops to interact, and single-molecule FRET studies reveal that folding proceeds via a discrete intermediate. Folding occurs in the absence of adenine ligand, but ligand binding stabilizes the folded structure by increasing the folding rate and decreasing the unfolding rate, and it lowers the magnesium ion concentration required to promote the loop-loop interaction. Individual aptamer molecules exhibit great heterogeneity in folding and unfolding rates, but this is reduced in the presence of adenine. In the full riboswitch, the adenine binding domain fails to fold because of conformational competition by the terminator stem. Thus, riboswitch function should depend on the relative rates of ligand binding and the transcriptional process.

The Speed of RNA Transcription and Metabolite Binding Kinetics Operate an FMN Riboswitch

Riboswitches are genetic control elements that usually reside in untranslated regions of messenger RNAs. These folded RNAs directly bind metabolites and undergo allosteric changes that modulate gene expression. A flavin mononucleotide (FMN)-dependent riboswitch from the ribDEAHT operon of Bacillus subtilis uses a transcription termination mechanism wherein formation of an RNA-FMN complex causes formation of an intrinsic terminator stem. We assessed the importance of RNA transcription speed and the kinetics of FMN binding to the nascent mRNA for riboswitch function. The riboswitch does not attain thermodynamic equilibrium with FMN before RNA polymerase needs to make a choice between continued transcription and transcription termination. Therefore, this riboswitch is kinetically driven, and functions more like a "molecular fuse." This reliance on the kinetics of ligand association and RNA polymerization speed might be common for riboswitches that utilize transcription termination mechanisms.

Temperature sensitivity caused by mutant release factor 1 is suppressed by mutations that affect 16S rRNA maturation.

To study the effect of slow termination on the protein synthesizing machinery, we isolated suppressors to a temperature-sensitive release factor 1 (RF1). Of 26 independent clones, five complementation groups have been identified, two of which are presented here. The first mutation disrupts a base pair in the transcription terminator stem for the rplM-rpsI operon, which encodes ribosomal proteins L13 and S9. We have found that this leads to readthrough of the terminator and that lower levels of transcript (compared to the results seen with the wild type) are found in the cell. This probably leads to decreased expression of the two proteins. The second mutation is a small deletion of the yrdC open reading frame start site, and it is not likely that the protein is expressed. Both mutant strains show an increased accumulation of 17S rRNA (immature 16S rRNA). Maturation of 16S rRNA is dependent on proper assembly of the ribosomal proteins, a process that is disturbed when proteins are missing. The function of the YrdC protein is not known, but it is able to bind to double-stranded RNA; therefore, we suggest that it is an assembly factor important for 30S subunit biogenesis. On the basis of our findings, we propose that lesser amounts of S9 or a lack of YrdC causes the maturation defect. We have shown that as a consequence of the maturation defect, fewer 70S ribosomes and polysomes are formed. This and other results suggest that it is the lowered concentration of functional ribosomes that suppresses the temperature sensitivity caused by the mutant RF1.

Adenine riboswitches and gene activation by disruption of a transcription terminator.

A class of riboswitches that recognizes guanine and discriminates against other purine analogs was recently identified. RNAs that carry the consensus sequence and structural features of guanine riboswitches are located in the 5' untranslated region (UTR) of numerous prokaryotic genes, where they control the expression of proteins involved in purine salvage and biosynthesis. We report that three representatives of this riboswitch class bind adenine with values for apparent dissociation constant (apparent K(d)) that are several orders of magnitude lower than those for binding guanine. Because preference for adenine is attributable to a single nucleotide substitution, the RNA most likely recognizes its ligand by forming a Watson-Crick base pair. In addition, the adenine riboswitch associated with the ydhL gene of Bacillus subtilis functions as a genetic 'on' switch, wherein adenine binding causes a structural rearrangement that precludes formation of an intrinsic transcription terminator stem.

Cis-acting sequences of Bacillus subtilis pyrG mRNA essential for regulation by antitermination.

Expression of the Bacillus subtilis pyrG gene, which encodes CTP synthetase, is repressed by cytidine nucleotides. Regulation involves a termination-antitermination mechanism acting at a transcription terminator located within the 5' untranslated pyrG leader sequence. Deletion and substitution mutagenesis of a series of pyrG'-lacZ transcriptional fusions integrated into the B. subtilis chromosome demonstrated that only the terminator stem-loop and two specific 4- to 6-nucleotide RNA sequences were required for derepression of pyrG by starvation for cytidine nucleotides. The first sequence, GGGC/U, comprises the first four nucleotides at the 5' end of the pyrG transcript, and the second, GCUCCC, forms the first six nucleotides of the 5' strand of the terminator stem. All of the nucleotides lying between the two required RNA sequences can be deleted without loss of regulation. We propose that an as-yet-unidentified regulatory protein binds to these two RNA segments and prevents termination of transcription in the pyrG leader region when intracellular CTP levels are low.

Primary structure requirements for in vivo activity and bidirectional function of the transcription terminator shared by the oppositely oriented skc/rel-orf1 genes of Streptococcus equisimilis H46A.

The region between the Streptococcus equisimilis streptokinase (skc) gene and the oppositely oriented rel-orf1 transcription unit contains only one termination site known to function bidirectionally in both the homologous host and in Escherichia coli. The terminator sequence is similar to other factor-independent terminators. Using two sets of point mutations that interrupt the hairpin-upstream oligo(dA) tract or the hairpin-downstream oligo(dT) tract, we examined the possible contribution of extended base pairing between the upstream rA and downstream rU residues to efficient termination and bidirectionality in both hosts, using terminator-cat reporter gene fusions in either polarity. The results show that interrupting the oligo(dA) tract preceding the hairpin has relatively little effect on terminator strength in either orientation in the homologous host, but abolishes termination in skc polarity in E. coli. Disruption of the hairpin-distal oligo(dT) tract inactivated the terminator in skc polarity in both hosts, had little effect on termination efficiency in rel-orf1 polarity in S. equisimilis, and also retained appreciable terminator activity in E. coli. In general, these alterations of the terminator sequence, together with additional mutations that reduce the spacing between the skc stop codon and the termination site or introduce a base substitution in the terminator stem, adversely affected the efficiency of termination to a greater extent in E. coli than in the homologous host. The disparity between the effects of certain mutations in the two hosts suggests that, in addition to thermodynamic properties, specific host factors, including RNA polymerase, contribute to terminator strength.