Cyclic di-GMP is a circular RNA dinucleotide that functions as a second messenger in diverse species of bacteria to trigger wide-ranging physiological changes, including cell differentiation, conversion between motile and biofilm lifestyles, and virulence gene expression. However, the mechanisms used by cyclic di-GMP to regulate gene expression have remained a mystery. We demonstrate that cyclic di-GMP in many bacterial species is sensed by a riboswitch class in mRNA that controls the expression of genes involved in numerous fundamental cellular processes. A variety of cyclic di-GMP regulons are revealed, including some riboswitches associated with virulence gene expression, pili formation, and flagellar organelle biosynthesis. In addition, sequences matching the consensus for cyclic di-GMP riboswitches are present in the genome of a bacteriophage.

Messenger RNAs are typically thought of as passive carriers of genetic information that are acted upon by protein- or small RNA-regulatory factors and by ribosomes during the process of translation. We report that the 5'-untranslated sequence of the Escherichia coli btuB mRNA assumes a more proactive role in metabolic monitoring and genetic control. The mRNA serves as a metabolite-sensing genetic switch by selectively binding coenzyme B(12) without the need for proteins. This binding event establishes a distinct RNA structure that is likely to be responsible for inhibition of ribosome binding and consequent reduction in synthesis of the cobalamin transport protein BtuB. This finding, along with related observations, supports the hypothesis that metabolic monitoring through RNA-metabolite interactions is a widespread mechanism of genetic control.

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...

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.