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.

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...

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A riboswitch within the 5' untranslated region (UTR) of the Bacillus subtilis pbuE mRNA binds adenine and related analogues in the absence of protein factors; excess adenine added to bacterial growth media triggers activation of a reporter gene that carries this riboswitch. To assess whether the riboswitch reaches thermodynamic equilibrium, or is operated by the kinetics of ligand binding and RNA transcription, we examined the detailed equilibrium and kinetic parameters for the complex formation between the aptamer domain of this riboswitch and the ligands adenine, 2-aminopurine (2AP), and 2,6-diaminopurine (DAP). Using a fluorescence-based assay, we have confirmed that adenine and 2AP have nearly equal binding affinity, with KD values for 2AP ranging from 250 nM to 3 microM at temperatures ranging from 15 to 35 degrees C, while DAP binds with much higher affinity. The association rate constant, however, favors adenine over DAP and 2AP by 3- and 10-fold, respectively, at 25 degrees C. Furthermore, the rate constants for adenine association and dissociation with the aptamer suggest that the pbuE riboswitch could be either kinetically or thermodynamically controlled depending upon the time scale of transcription and external variables such as temperature. We cite data that suggest kinetic control under certain conditions and illustrate with a model calculation how the system can switch between kinetic and equilibrium control. These findings further support the hypothesis that many riboswitches rely on the kinetics of ligand binding and the speed of RNA transcription, rather than simple ligand affinity, to establish the concentration of metabolite needed to trigger riboswitch function.

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