A riboswitch selective for the queuosine precursor preQ1 contains an unusually small aptamer domain.

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.

Ligand binding and gene control characteristics of tandem riboswitches in Bacillus anthracis.

Most riboswitches are composed of a single metabolite-binding aptamer and a single expression platform that function together to regulate genes in response to changing metabolite concentrations. In rare instances, two aptamers or sometimes two complete riboswitches reside adjacent to each other in untranslated regions (UTRs) of mRNAs. We have examined an example of a tandem riboswitch in the Gram-positive bacterium Bacillus anthracis that includes two complete riboswitches for thiamine pyrophosphate (TPP). Unlike other complex riboswitch systems described recently, tandem TPP riboswitches do not exhibit cooperative ligand binding and do not detect two different types of metabolites. In contrast, both riboswitches respond independently to TPP and are predicted to function in concert to mimic the more "digital" gene control outcome observed when two aptamers bind ligands cooperatively. Our findings further demonstrate that simple gene control elements made only of RNA can be assembled in different architectures to yield more complex gene control outcomes.

Tandem riboswitch architectures exhibit complex gene control functions.

Riboswitches are structured RNAs typically located in the 5' untranslated regions of bacterial mRNAs that bind metabolites and control gene expression. Most riboswitches sense one metabolite and function as simple genetic switches. However, we found that the 5' region of the Bacillus clausii metE messenger RNA includes two riboswitches that respond to S-adenosylmethionine and coenzyme B12. This tandem arrangement yields a composite gene control system that functions as a two-input Boolean NOR logic gate. These findings and the discovery of additional tandem riboswitch architectures reveal how simple RNA elements can be assembled to make sophisticated genetic decisions without involving protein factors.

Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria.

BACKGROUND: Riboswitches are RNA elements in the 5' untranslated leaders of bacterial mRNAs that directly sense the levels of specific metabolites with a structurally conserved aptamer domain to regulate expression of downstream genes. Riboswitches are most common in the genomes of low GC Gram-positive bacteria (for example, Bacillus subtilis contains examples of all known riboswitches), and some riboswitch classes seem to be restricted to this group. RESULTS: We used comparative sequence analysis and structural probing to identify five RNA elements (serC, speF, suhB, ybhL, and metA) that reside in the intergenic regions of Agrobacterium tumefaciens and many other alpha-proteobacteria. One of these, the metA motif, is found upstream of methionine biosynthesis genes and binds S-adenosylmethionine (SAM). This natural aptamer most likely functions as a SAM riboswitch (SAM-II) with a consensus sequence and structure that is distinct from the class of SAM riboswitches (SAM-I) predominantly found in Gram-positive bacteria. The minimal functional SAM-II aptamer consists of fewer than 70 nucleotides, which form a single stem and a pseudoknot. Despite its simple architecture and lower affinity for SAM, the SAM-II aptamer strongly discriminates against related compounds. CONCLUSION: SAM-II is the only metabolite-binding riboswitch class identified so far that is not found in Gram-positive bacteria, and its existence demonstrates that biological systems can use multiple RNA structures to sense a single chemical compound. The two SAM riboswitches might be 'RNA World' relics that were selectively retained in certain bacterial lineages or new motifs that have emerged since the divergence of the major bacterial groups.

Stereoselective synthesis of trans-threo-trans-oligopyrrolidines: potential agents for RNA cleavage.

The 2,5-trans-substituted oligopyrrolidines constitute a promising class of novel RNA-binding agents as well as potential building blocks for artificial anion channels. A convergent synthesis of terpyrrolidine 1 and pyrrolidino-THF-pyrrolidine 2 is reported, relying upon convergent coupling of 2,5-trans-pyrrolidinecarboxaldehydes through bridging alkyne units under Felkin-Anh control and subsequent closure of the central ring. After complete deprotection, the free polyamine products were isolated in excellent yield and purity. Crystal structure analyses of a terpyrrolidine and a pyrrolidino-THF-pyrrolidine documented their helical privileged conformations. The compounds were then screened for RNA cleavage activity. Unlike the only weakly active simple polyamines, p-nitrosulfonamide 33 was found to induce cleavage at mM concentrations under physiologically relevant conditions.