ABSTRACT
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.
Subject(s)
Aptamers, Nucleotide/chemistry , Bacillus subtilis/genetics , Nucleoside Q/metabolism , Pyrimidinones/metabolism , Pyrroles/metabolism , Regulatory Sequences, Ribonucleic Acid , 5' Untranslated Regions/genetics , Aptamers, Nucleotide/genetics , Base Pairing/genetics , Base Sequence , Conserved Sequence , Dialysis , Gene Expression Regulation, Bacterial , Genes, Bacterial , Molecular Sequence Data , Nucleoside Q/chemistry , Phylogeny , Pyrimidinones/chemistry , Pyrroles/chemistry , RNA, Bacterial/chemistry , RNA, Bacterial/geneticsABSTRACT
The glmS ribozyme is a riboswitch class that occurs in certain Gram-positive bacteria, where it resides within mRNAs encoding glucosamine 6-phosphate synthase. Members of this self-cleaving ribozyme class rapidly catalyze RNA transesterification upon binding GlcN6P, and genetic evidence suggests that this cleavage event is important for down-regulating GlmS protein expression. In this report, we present a refined secondary structure model of the glmS ribozyme and determine the importance of a conserved pseudoknot structure for optimal ribozyme function. Analyses of deletion constructs demonstrate that the pseudoknot, together with other structural elements, permits the ribozyme to achieve maximum rate constants for RNA cleavage at physiologically relevant Mg2+ concentrations. In addition, we show that substantial rate enhancements are supported by an exchange-inert cobalt (III) complex and by molar concentrations of monovalent ions. Our findings indicate that the glmS ribozyme forms a complex structure to employ catalytic strategies that do not require the direct participation of divalent metal ions.
Subject(s)
Bacterial Proteins/genetics , Cations, Divalent , Metals/metabolism , RNA, Bacterial/genetics , Bacillus cereus/enzymology , Bacillus cereus/genetics , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Base Sequence , Kinetics , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Bacterial/chemistry , Sequence Homology, Nucleic AcidABSTRACT
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, ydaO/yuaA, ykkC/yxkD, ykoK, and yybP/ykoY). 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 Gram-positive 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.
