Most biological catalysts are made of protein; however, eight classes of natural ribozymes have been discovered that catalyse fundamental biochemical reactions. The central functions of ribozymes in modern organisms support the hypothesis that life passed through an 'RNA world' before the emergence of proteins and DNA. We have identified a new class of ribozymes that cleaves the messenger RNA of the glmS gene in Gram-positive bacteria. The ribozyme is activated by glucosamine-6-phosphate (GlcN6P), which is the metabolic product of the GlmS enzyme. Additional data indicate that the ribozyme serves as a metabolite-responsive genetic switch that represses the glmS gene in response to rising GlcN6P concentrations. These findings demonstrate that ribozyme switches may have functioned as metabolite sensors in primitive organisms, and further suggest that modern cells retain some of these ancient genetic control systems.

The expression of certain genes involved in fundamental metabolism is regulated by metabolite-binding ''riboswitch'' elements embedded within their corresponding mRNAs. We have identified at least six additional elements within the Bacillus subtilis genome that exhibit characteristics of riboswitch function (glmS, gcvT, ydaO͞yuaA, ykkC͞yxkD, ykoK, and yybP͞ykoY). These motifs exhibit extensive sequence and secondary-structure conservation among many bacterial species and occur upstream of related genes. The element located upstream of the glmS gene in Grampositive organisms functions as a metabolite-dependent ribozyme that responds to glucosamine-6-phosphate. Other motifs form complex folded structures when transcribed as RNA molecules and carry intrinsic terminator structures. These findings indicate that riboswitches serve as a major genetic regulatory mechanism for the control of metabolic genes in many microbial species. R iboswitches are highly structured domains within mRNAs that precisely sense metabolites and control gene expression (1). These RNA elements are capable of binding to a variety of target compounds and subsequently modulating transcription and translation with performance characteristics that are similar to those of protein genetic factors. Typically, each riboswitch is composed of a conserved metabolite-binding domain (aptamer) located upstream of a variable sequence region (expression platform) that dictates the level of gene expression. Allosteric changes brought about by metabolite binding to the aptamer are harnessed by the expression platform to modulate the expression of the adjacent gene or operon. Riboswitches are versatile genetic control elements. In some instances, both transcription and translation control are used by the same aptamer class in the same prokaryotic organism (e.g., see ref.2). Evidence also shows that riboswitches can use mRNA-processing events to modulate gene expression (3, 4).The various metabolites that are detected by known riboswitches are of fundamental importance to living systems (5). On this basis, we have speculated that modern riboswitches might be the remaining representatives of an ancient metabolitemonitoring system that was present in the RNA World (5-9). The wide distribution of some riboswitch classes among microbes (e.g., see refs. 5 and 9-14) and the presence of metabolitebinding RNA domains in eukaryotes (4) support this hypothesis. Each of the seven classes of riboswitches reported (1, 5) was examined for metabolite-binding function because published genetic evidence showed that these elements were important for genetic control. Because the regulation of many metabolism genes has not been characterized in detail, it is possible that numerous other metabolite-binding RNA motifs exist in nature.The riboswitches known to be present in prokaryotes are typically located in noncoding or intergenic regions (IGRs). Therefore, the examination of unusually long IGRs for indications of conserved sequence and secondary-structure elements should yield new...

The cellular concentrations of certain metabolites are assiduously monitored to achieve appropriate levels of gene expression. While proteins have long been known to act as sensors in this capacity, metabolite-binding RNAs, or riboswitches, also play an important role. More than 20 distinct classes of riboswitches have been identified to date, and insights to the molecular recognition strategies of a significant subset of these have been provided by detailed structural studies. This diverse set of metabolite-sensing RNAs is found to exploit a variety of distinct mechanisms to regulate genes that are fundamental to metabolism.

Most riboswitches are metabolite-binding RNA structures located in bacterial messenger RNAs where they control gene expression. We have discovered a riboswitch class in many bacterial and archaeal species whose members are selectively triggered by fluoride but reject other small anions, including chloride. These fluoride riboswitches activate expression of genes that encode putative fluoride transporters, enzymes that are known to be inhibited by fluoride, and additional proteins of unknown function. Our findings indicate that most organisms are naturally exposed to toxic levels of fluoride and that many species use fluoride-sensing RNAs to control the expression of proteins that alleviate the deleterious effects of this anion.

Ribozymes are noncoding RNAs that promote chemical transformations with rate enhancements approaching those of protein enzymes. Although ribozymes are likely to have been abundant during the RNA world era, only ten classes are known to exist among contemporary organisms. We report the discovery and analysis of an additional self-cleaving ribozyme class, called twister, which is present in many species of bacteria and eukarya. Nearly 2700 twister ribozymes were identified that conform to a secondary structure consensus that is small yet complex, with three stems conjoined by internal and terminal loops. Two pseudoknots provide tertiary structure contacts that are critical for catalytic activity. The twister ribozyme motif provides another example of a natural RNA catalyst and calls attention to the potentially varied biological roles of this and other classes of widely distributed self-cleaving RNAs.

We applied a computational pipeline based on comparative genomics to bacteria, and identified 22 novel candidate RNA motifs. We predicted six to be riboswitches, which are mRNA elements that regulate gene expression on binding a specific metabolite. In separate studies, we confirmed that two of these are novel riboswitches. Three other riboswitch candidates are upstream of either a putative transporter gene in the order Lactobacillales, citric acid cycle genes in Burkholderiales or molybdenum cofactor biosynthesis genes in several phyla. The remaining riboswitch candidate, the widespread Genes for the Environment, for Membranes and for Motility (GEMM) motif, is associated with genes important for natural competence in Vibrio cholerae and the use of metal ions as electron acceptors in Geobacter sulfurreducens. Among the other motifs, one has a genetic distribution similar to a previously published candidate riboswitch, ykkC/yxkD, but has a different structure. We identified possible non-coding RNAs in five phyla, and several additional cis-regulatory RNAs, including one in ε-proteobacteria (upstream of purD, involved in purine biosynthesis), and one in Cyanobacteria (within an ATP synthase operon). These candidate RNAs add to the growing list of RNA motifs involved in multiple cellular processes, and suggest that many additional RNAs remain to be discovered.

A previous bioinformatics-based search for riboswitches yielded several candidate motifs in eubacteria. One of these motifs commonly resides in the 5' untranslated regions of genes involved in the biosynthesis of queuosine (Q), a hypermodified nucleoside occupying the anticodon wobble position of certain transfer RNAs. Here we show that this structured RNA is part of a riboswitch selective for 7-aminomethyl-7-deazaguanine (preQ(1)), an intermediate in queuosine biosynthesis. Compared with other natural metabolite-binding RNAs, the preQ(1) aptamer appears to have a simple structure, consisting of a single stem-loop and a short tail sequence that together are formed from as few as 34 nucleotides. Despite its small size, this aptamer is highly selective for its cognate ligand in vitro and has an affinity for preQ(1) in the low nanomolar range. Relatively compact RNA structures can therefore serve effectively as metabolite receptors to regulate gene expression.

The speed at which RNA molecules decompose is a critical determinant of many biological processes, including those directly involved in the storage and expression of genetic information. One mechanism for RNA cleavage involves internal phosphoester transfer, wherein the 2-oxygen atom carries out an S N 2-like nucleophilic attack on the adjacent phosphorus center (transesterification). In this article, we discuss fundamental principles of RNA transesterification and define a conceptual framework that can be used to assess the catalytic power of enzymes that cleave RNA. We deduce that certain ribozymes and deoxyribozymes, like their protein enzyme counterparts, can bring about enormous rate enhancements.