Madeline E. Sherlock

Riboswitch diversity and distribution

Riboswitches are commonly used by bacteria to detect a variety of metabolites and ions to regulate gene expression. To date, nearly 40 different classes of riboswitches have been discovered, experimentally validated, and modeled at atomic resolution in complex with their cognate ligands. The research findings produced since the first riboswitch validation reports in 2002 reveal that these noncoding RNA domains exploit many different structural features to create binding pockets that are extremely selective for their target ligands. Some riboswitch classes are very common and are present in bacteria from nearly all lineages, whereas others are exceedingly rare and appear in only a few species whose DNA has been sequenced. Presented herein are the consensus sequences, structural models, and phylogenetic distributions for all validated riboswitch classes. Based on our findings, we predict that there are potentially many thousands of distinct bacterial riboswitch classes remaining to be discovered, but that the rarity of individual undiscovered classes will make it increasingly difficult to find additional examples of this RNA-based sensory and gene control mechanism.

Metabolism of Free Guanidine in Bacteria Is Regulated by a Widespread Riboswitch Class

The guanidyl moiety is a component of fundamental metabolites, including the amino acid arginine, the energy carrier creatine, and the nucleobase guanine. Curiously, reports regarding the importance of free guanidine in biology are sparse, and no biological receptors that specifically recognize this compound have been previously identified. We report that many members of the ykkC motif RNA, the longest unresolved riboswitch candidate, naturally sense and respond to guanidine. This RNA is found throughout much of the bacterial domain of life, where it commonly controls the expression of proteins annotated as urea carboxylases and multidrug efflux pumps. Our analyses reveal that these proteins likely function as guanidine carboxylases and guanidine transporters, respectively. Furthermore, we demonstrate that bacteria are capable of endogenously producing guanidine. These and related findings demonstrate that free guanidine is a biologically relevant compound, and several gene families that can alleviate guanidine toxicity exist.

Effect of loop sequence and loop length on the intrinsic fluorescence of G-quadruplexes.

Guanine quadruplex structures (GQSs) exhibit unique spectroscopic features, including an inverse melting profile at 295 nm, distinctive circular dichroism features, and intrinsic fluorescence. Herein, we investigate effects of loop sequence and loop length on the intrinsic fluorescence of 13 DNA GQSs. We report label-free fluorescence enhancements upon intramolecular GQS formation of up to 16-fold and a shift in the emission maximum to the visible portion of the spectrum. Effects can be understood in the context of available nuclear magnetic resonance GQS structures. The intrinsic fluorescence of GQSs may be useful for nucleic acid studies and for the development of label-free detection methods.

Bioinformatic analysis of riboswitch structures uncovers variant classes with altered ligand specificity

Significance In the 15 y since metabolite-binding riboswitches were first experimentally validated, only 4 examples of riboswitch classes with altered specificity have been confirmed by experiments out of ∼30 distinct structural architectures. In contrast, evolutionary changes in ligand specificity of proteins are routinely reported. To further investigate the propensity for natural adaptation of riboswitch specificity, we developed a structural bioinformatics method to systematically search for variant riboswitches with altered ligand recognition. This search method yielded evidence for altered specificity within five riboswitch classes, including validation of a second riboswitch class that senses 2′-deoxyguanosine. Riboswitches are RNAs that form complex, folded structures that selectively bind small molecules or ions. As with certain groups of protein enzymes and receptors, some riboswitch classes have evolved to change their ligand specificity. We developed a procedure to systematically analyze known riboswitch classes to find additional variants that have altered their ligand specificity. This approach uses multiple-sequence alignments, atomic-resolution structural information, and riboswitch gene associations. Among the discoveries are unique variants of the guanine riboswitch class that most tightly bind the nucleoside 2′-deoxyguanosine. In addition, we identified variants of the glycine riboswitch class that no longer recognize this amino acid, additional members of a rare flavin mononucleotide (FMN) variant class, and also variants of c-di-GMP-I and -II riboswitches that might recognize different bacterial signaling molecules. These findings further reveal the diverse molecular sensing capabilities of RNA, which highlights the potential for discovering a large number of additional natural riboswitch classes.

Biochemical Validation of a Second Guanidine Riboswitch Class in Bacteria

Recently, it was determined that representatives of the riboswitch candidates called ykkC and ykkC-III directly bind free guanidine. Guanidine-binding ykkC motif RNAs, now renamed guanidine-I riboswitches, were demonstrated to commonly regulate the expression of genes encoding guanidine carboxylases, as well as others encoding guanidine efflux proteins such as EmrE and SugE. Likewise, genes encoding similar efflux proteins are associated with ykkC-III motif RNAs, which have now been renamed guanidine-III riboswitches. Prior to the validation of guanidine as the ligand for these newly established riboswitch classes, another RNA motif was discovered by comparative genomic analysis and termed mini-ykkC because of its small size and gene associations similar to those of the original ykkC motif. It was hypothesized that these distinct RNA structures might respond to the same ligand. However, the small size and repetitive nature of mini-ykkC RNAs suggested that it might respond to ligand via the action of a protein factor. Herein, we demonstrate that, despite its extremely simple architecture, mini-ykkC motif RNAs constitute a distinct class of guanidine-sensing RNAs, called guanidine-II riboswitches. Surprisingly, each of the two stem-loop structures that comprise the mini-ykkC motif appears to directly bind free guanidine in a cooperative manner. These findings reveal that bacteria make extensive use of diverse guanidine-responsive riboswitches to overcome the toxic effects of this compound.

Detection of 224 candidate structured RNAs by comparative analysis of specific subsets of intergenic regions

Abstract The discovery of structured non-coding RNAs (ncRNAs) in bacteria can reveal new facets of biology and biochemistry. Comparative genomics analyses executed by powerful computer algorithms have successfully been used to uncover many novel bacterial ncRNA classes in recent years. However, this general search strategy favors the discovery of more common ncRNA classes, whereas progressively rarer classes are correspondingly more difficult to identify. In the current study, we confront this problem by devising several methods to select subsets of intergenic regions that can concentrate these rare RNA classes, thereby increasing the probability that comparative sequence analysis approaches will reveal their existence. By implementing these methods, we discovered 224 novel ncRNA classes, which include ROOL RNA, an RNA class averaging 581 nt and present in multiple phyla, several highly conserved and widespread ncRNA classes with properties that suggest sophisticated biochemical functions and a multitude of putative cis-regulatory RNA classes involved in a variety of biological processes. We expect that further research on these newly found RNA classes will reveal additional aspects of novel biology, and allow for greater insights into the biochemistry performed by ncRNAs.

Decrease in RNA folding cooperativity by deliberate population of intermediates in RNA G-quadruplexes

Keeping a broad (RNA) perspective: conventional biochemical detection systems only have a 100-fold linear response range. The range of potassium concentrations detected by an RNA G-quadruplex sequence can be broadened by intentionally populating multiple intermediate folding states. The folding of the RNA G-quadruplexes was monitored by both circular dichroism and intrinsic fluorescence spectroscopy.

A hybridization-based approach for quantitative and low-bias single-stranded DNA ligation.

Single-stranded DNA (ssDNA) ligation is a crucial step in many biochemical assays. Efficient ways of carrying out this reaction, however, are lacking. We show here that existing ssDNA ligation methods suffer from slow kinetics, poor yield, and severe nucleotide preference. To resolve these issues, we introduce a hybridization-based strategy that provides efficient and low-bias ligation of ssDNA. Our method uses a hairpin DNA to hybridize to any incoming acceptor ssDNA with low bias, with ligation of these strands mediated by T4 DNA ligase. This technique potentially can be applied in protocols that require ligation of ssDNA, including ligation-mediated polymerase chain reaction (LMPCR) and complementary DNA (cDNA) library construction.

Tandem riboswitches form a natural Boolean logic gate to control purine metabolism in bacteria

Gene control systems sometimes interpret multiple signals to set the expression levels of the genes they regulate. In rare instances, ligand-binding riboswitch aptamers form tandem arrangements to approximate the function of specific two-input Boolean logic gates. Here, we report the discovery of riboswitch aptamers for phosphoribosyl pyrophosphate (PRPP) that naturally exist either in singlet arrangements, or occur in tandem with guanine aptamers. Tandem guanine-PRPP aptamers can bind the target ligands, either independently or in combination, to approximate the function expected for an IMPLY Boolean logic gate to regulate transcription of messenger RNAs for de novo purine biosynthesis in bacteria. The existence of sophisticated all-RNA regulatory systems that sense two ancient ribonucleotide derivatives to control synthesis of RNA molecules supports the hypothesis that RNA World organisms could have managed a complex metabolic state without the assistance of protein regulatory factors.

Riboswitches for the alarmone ppGpp expand the collection of RNA-based signaling systems

Significance Bacteria and other organisms make extensive use of signaling molecules that are derived from ribonucleotides or their derivatives. Previously, five riboswitch classes had been discovered that sense the four RNA-derived signaling molecules: c-di-GMP, c-di-AMP, c-AMP-GMP, and ZTP. We now report the discovery and biochemical validation of bacterial riboswitches for the widespread alarmone guanosine tetraphosphate (ppGpp), which signals metabolic and physiological adaptations to starvation. These findings expand the number of natural partnerships between riboswitches and ribonucleotide-like signaling molecules, and provide RNA-based sensors for detecting ppGpp production in cells. Riboswitches are noncoding portions of certain mRNAs that bind metabolite, coenzyme, signaling molecule, or inorganic ion ligands and regulate gene expression. Most known riboswitches sense derivatives of RNA monomers. This bias in ligand chemical composition is consistent with the hypothesis that widespread riboswitch classes first emerged during the RNA World, which is proposed to have existed before proteins were present. Here we report the discovery and biochemical validation of a natural riboswitch class that selectively binds guanosine tetraphosphate (ppGpp), a widespread signaling molecule and bacterial “alarmone” derived from the ribonucleotide GTP. Riboswitches for ppGpp are predicted to regulate genes involved in branched-chain amino acid biosynthesis and transport, as well as other gene classes that previously had not been implicated to be part of its signaling network. This newfound riboswitch–alarmone partnership supports the hypothesis that prominent RNA World signaling pathways have been retained by modern cells to control key biological processes.