Diversity and modularity of tyrosine-accepting tRNA-like structures.

Certain positive-sense single-stranded RNA viruses contain elements at their 3' termini that structurally mimic tRNAs. These tRNA-like structures (TLSs) are classified based on which amino acid is covalently added to the 3' end by host aminoacyl-tRNA synthetase. Recently, a cryoEM reconstruction of a representative tyrosine-accepting tRNA-like structure (TLS) from brome mosaic virus (BMV) revealed a unique mode of recognition of the viral anticodon-mimicking domain by tyrosyl-tRNA synthetase.

A conserved class of viral RNA structures regulate translation reinitiation through dynamic ribosome interactions.

Certain viral RNAs encode proteins downstream of the main protein coding region, expressed through "termination-reinitiation" events, dependent on RNA structure. RNA elements located upstream of the first stop codon within these viral mRNAs bind the ribosome, preventing ribosome recycling and inducing reinitiation. We used bioinformatic methods to identify new examples of viral reinitiation-stimulating RNAs and experimentally verified their secondary structure and function.

Principles, mechanisms, and biological implications of translation termination-reinitiation.

The gene expression pathway from DNA sequence to functional protein is not as straightforward as simple depictions of the central dogma might suggest. Each step is highly regulated, with complex and only partially understood molecular mechanisms at play. Translation is one step where the "one gene-one protein" paradigm breaks down, as often a single mature eukaryotic mRNA leads to more than one protein product.

Architectures and complex functions of tandem riboswitches.

Riboswitch architectures that involve the binding of a single ligand to a single RNA aptamer domain result in ordinary dose-response curves that require approximately a 100-fold change in ligand concentration to cover nearly the full dynamic range for gene regulation. However, by using multiple riboswitches or aptamer domains in tandem, these ligand-sensing structures can produce additional, complex gene control outcomes. In the current study, we have computationally searched for tandem riboswitch architectures in bacteria to provide a more complete understanding of the diverse biological and biochemical functions of gene control elements that are made exclusively of RNA.

A viral RNA hijacks host machinery using dynamic conformational changes of a tRNA-like structure.

Viruses require multifunctional structured RNAs to hijack their host’s biochemistry, but their mechanisms can be obscured by the difficulty of solving conformationally dynamic RNA structures. Using cryo–electron microscopy (cryo-EM), we visualized the structure of the mysterious viral transfer RNA (tRNA)–like structure (TLS) from the brome mosaic virus, which affects replication, translation, and genome encapsidation. Structures in isolation and those bound to tyrosyl-tRNA synthetase (TyrRS) show that this ~55-kilodalton purported tRNA mimic undergoes large conformational rearrangements to bind TyrRS in a form that differs substantially from that of tRNA.

An expanded class of histidine-accepting viral tRNA-like structures.

Structured RNA elements are common in the genomes of RNA viruses, often playing critical roles during viral infection. Some viral RNA elements use forms of tRNA mimicry, but the diverse ways this mimicry can be achieved are poorly understood. Histidine-accepting tRNA-like structures (TLS) are examples found at the 3' termini of some positive-sense single-stranded RNA (+ssRNA) viruses where they interact with several host proteins, induce histidylation of the RNA genome, and facilitate processes important for infection, to include genome replication.

Structural diversity and phylogenetic distribution of valyl tRNA-like structures in viruses.

Viruses commonly use specifically folded RNA elements that interact with both host and viral proteins to perform functions important for diverse viral processes. Examples are found at the 3' termini of certain positive-sense ssRNA virus genomes where they partially mimic tRNAs, including being aminoacylated by host cell enzymes. Valine-accepting tRNA-like structures (TLS) are an example that share some clear homology with canonical tRNAs but have several important structural differences.

Former orphan riboswitches reveal unexplored areas of bacterial metabolism, signaling, and gene control processes.

Comparative sequence analyses have been used to discover numerous classes of structured noncoding RNAs, some of which are riboswitches that specifically recognize small-molecule or elemental ion ligands and influence expression of adjacent downstream genes. Determining the correct identity of the ligand for a riboswitch candidate typically is aided by an understanding of the genes under its regulatory control. Riboswitches whose ligands were straightforward to identify have largely been associated with well-characterized metabolic pathways, such as coenzyme or amino acid biosynthesis.

Variant Bacterial Riboswitches Associated with Nucleotide Hydrolase Genes Sense Nucleoside Diphosphates.

The ykkC RNA motif was a long-standing orphan riboswitch candidate that has recently been proposed to encompass at least five distinct bacterial riboswitch classes. Most ykkC RNAs belong to the subtype 1 group, which are guanidine-I riboswitches that regulate the expression of guanidine-specific carboxylase and transporter proteins. The remaining ykkC RNAs have been organized into at least four major categories called subtypes 2a-2d.

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

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.

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.

Challenges of ligand identification for the second wave of orphan riboswitch candidates.

Orphan riboswitch candidates are noncoding RNA motifs whose representatives are believed to function as genetic regulatory elements, but whose target ligands have yet to be identified. The study of certain orphans, particularly classes that have resisted experimental validation for many years, has led to the discovery of important biological pathways and processes once their ligands were identified. Previously, we highlighted details for four of the most common and intriguing orphan riboswitch candidates.

SAM-VI RNAs selectively bind S-adenosylmethionine and exhibit similarities to SAM-III riboswitches.

Five distinct riboswitch classes that regulate gene expression in response to the cofactor S-adenosylmethionine (SAM) or its metabolic breakdown product S-adenosylhomocysteine (SAH) have been reported previously. Collectively, these SAM- or SAH-sensing RNAs constitute the most abundant collection of riboswitches, and are found in nearly every major bacterial lineage. Here, we report a potential sixth member of this pervasive riboswitch family, called SAM-VI, which is predominantly found in Bifidobacterium species.

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

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.

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.

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

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.

The Biology of Free Guanidine As Revealed by Riboswitches.

The experimental validation of three distinct riboswitch classes has revealed that many bacterial cells naturally produce guanidine, and that living systems have evolved a variety of genes involved in the metabolism and transport of this toxic metabolite. There are numerous biochemical curiosities and mysteries that spring from these advances, which will make for interesting research topics in the coming years.

Biochemical Validation of a Third Guanidine Riboswitch Class in Bacteria.

Recently, it was determined that representatives of the riboswitch candidates called ykkC and mini-ykkC directly bind free guanidine. These riboswitches regulate the expression of genes whose protein products are implicated in overcoming the toxic effects of this ligand. Thus, the relevant ykkC motif and mini-ykkC motif RNAs have been classified as guanidine-I and guanidine-II riboswitch RNAs, respectively.

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.

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.

Steady-State and Time-Resolved Studies into the Origin of the Intrinsic Fluorescence of G-Quadruplexes.

Stretches of guanines in DNA and RNA can fold into guanine quadruplex structures (GQSs). These structures protect telomeres in DNA and regulate gene expression in RNA. GQSs have an intrinsic fluorescence that is sensitive to different parameters, including loop sequence and length.

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.

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.

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.