By using a structure-based fluorescence spectroscopic approach, we have examined the folding of an adenine-responsive riboswitch that regulates translation initiation. We observed adaptive recognition of the ligand for the aptamer domain of adenosine deaminase (add) mRNA from Vibrio vulnificus, and revealed pronounced conformational changes even in the preorganized loop-loop region that is distant from the binding site. Importantly, the full-length riboswitch domain, which has a potential translational repressor stem is able to form a binary complex with adenine, and does not act as a folding trap to inhibit binding. The aptamer that is extended by the expression platform therefore remains fully responsive to its ligand; this is in contrast to the previously investigated pbuE A-riboswitch, which becomes trapped in a nonresponsive terminator fold. Consequently, the latter must employ complex response mechanisms, such as operating in kinetic-control mode or using transcriptional pausing, to provide time for the aptamer portion to fold and to bind. The different behavior of the riboswitches can be rationalized by their distinct sequence interface between the aptamer and expression platform. For the add A-riboswitch, our data suggest a thermodynamically driven response mechanism.

Riboswitches are genetic control elements within non-coding regions of mRNA. They consist of a metabolite-sensitive aptamer and an adjoining expression platform. Here, we describe ligand-induced folding of a thiamine pyrophosphate (TPP) responsive riboswitch from Escherichia coli thiM mRNA, using chemically labeled variants. Referring to a recent structure determination of the TPP/aptamer complex, each variant was synthesized with a single 2-aminopurine (AP) nucleobase replacement that was selected to monitor formation of tertiary interactions of a particular region during ligand binding in real time by fluorescence experiments. We have determined the rate constants for conformational adjustment of the individual AP sensors. From the 7-fold differentiation of these constants, it can be deduced that tertiary contacts between the two parallel helical domains (P2/J3-2/P3/L3 and P4/P5/L5) that grip the ligand's ends in two separate pockets, form significantly faster than the function-critical three-way junction with stem P1 fully developed. Based on these data, we characterize the process of ligand binding by an induced fit of the RNA and propose a folding model of the TPP riboswitch aptamer. For the full-length riboswitch domain and for shorter constructs that represent transcriptional intermediates, we have additionally evaluated ligand-induced folding via AP-modified variants and provide insights into the sequential folding pathway that involves a finely balanced equilibrium of secondary structures.

The ribosomal peptidyl transferase center is a ribozyme catalyzing peptide bond synthesis in all organisms. We applied a novel modified nucleoside interference approach to identify functional groups at 9 universally conserved active site residues. Owing to their immediate proximity to the chemical center, the 23S rRNA nucleosides A2451, U2506 and U2585 were of particular interest. Our study ruled out U2506 and U2585 as contributors of vital chemical groups for transpeptidation. In contrast the ribose 2'-OH of A2451 was identified as the prime ribosomal group with potential functional importance. This 2'-OH renders almost full catalytic power to the ribosome even when embedded into an active site of six neighboring 2'-deoxyribose nucleosides. These data highlight the unique functional role of the A2451 2'-OH for peptide bond synthesis among all other functional groups at the ribosomal peptidyl transferase active site.

Small non-coding RNAs (sRNAs) are an emerging class of post-transcriptional regulators of bacterial gene expression. To study sRNAs and their potential protein interaction partners, it is desirable to purify sRNAs from cells in their native form. Here, we used RNA-based affinity chromatography to purify sRNAs following their expression as aptamer-tagged variants in vivo. To this end, we developed a family of plasmids to express sRNAs with any of three widely used aptamer sequences (MS2, boxB, eIF4A), and systematically tested how the aptamer tagging impacted on intracellular accumulation and target regulation of the Salmonella GcvB, InvR or RybB sRNAs. In addition, we successfully tagged the chromosomal rybB gene with MS2 to observe that RybB-MS2 is fully functional as an envelope stress-induced repressor of ompN mRNA following induction of sigmaE. We further demonstrate that the common sRNA-binding protein, Hfq, co-purifies with MS2-tagged sRNAs of Salmonella. The presented affinity purification strategy may facilitate the isolation of in vivo assembled sRNA–protein complexes in a wide range of bacteria.

The derivatization of nucleic acids with selenium is a new and highly promising approach to facilitate their three-dimensional structure determination by X-ray crystallography. Here, we report a comprehensive study on the chemical and enzymatic syntheses of RNAs containing 2'-methylseleno (2'-Se-methyl) nucleoside labels. Our approach includes the first synthesis of an appropriate purine nucleoside phosphoramidite building block. Most importantly, a substantially changed RNA solid-phase synthesis cycle, comprising treatment with threo-1,4-dimercapto-2,3-butanediol (DTT) after the oxidation step, is required for a reliable strand elongation. This novel operation allows for the chemical syntheses of multiple Se-labeled RNAs in sizes that can typically be achieved only for nonmodified RNAs. In combination with enzymatic ligation, biologically important RNA targets become accessible for crystallography. Exemplarily, this has been demonstrated for the Diels-Alder ribozyme and the add adenine riboswitch sequences. We point out that the approach documented here has been the chemical basis for the very recent structure determination of the Diels-Alder ribozyme which represents the first novel RNA fold that has been solved via its Se-derivatives.

A precise tertiary structure must be adopted to allow the function of many RNAs in cells. Accordingly, increasing resources have been devoted to the elucidation of RNA structures and the folding of RNAs. 2-Aminopurine (2AP), a fluorescent nucleobase analogue, can be substituted in strategic positions of DNA or RNA molecules to act as site-specific probe to monitor folding and folding dynamics of nucleic acids. Recent studies further demonstrated the potential of 2AP modifications in the assessment of folding kinetics during ligand-induced secondary and tertiary RNA structure rearrangements. However, an efficient way to unambiguously identify reliable positions for 2AP sensors is as yet unavailable and would represent a major asset, especially in the absence of crystallographic or NMR structural data for a target molecule. We report evidence of a novel and direct correlation between the 2'-OH flexibility of nucleotides, observed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probing and the fluorescence response following nucleotide substitutions by 2AP. This correlation leads to a straightforward method, using SHAPE probing with benzoyl cyanide, to select appropriate nucleotide sites for 2AP substitution. This clear correlation is presented for three model RNAs of biological significance: the SAM-II, adenine (addA), and preQ(1) class II (preQ(1)cII) riboswitches.

Helicobacter pylori, one of the most prevalent human pathogens, used to be thought to lack small regulatory RNAs (sRNAs) which are otherwise considered abundant in all bacteria. However, our recent analysis of the primary transcriptome of H. pylori discovered an unexpectedly large number of sRNAs, and suggested that this model organism also uses riboregulation to control the expression of its genes. Nonetheless, whereas most enterobacterial sRNAs require the RNA chaperone Hfq for function, Epsilonproteobacteria including H. pylori seem to have no Hfq homologue, which prompted us to search for other auxiliary proteins in sRNA-mediated regulation. Therefore, we have developed two orthogonal methods to isolate and investigate in vivo and in vitro assembled RNA-protein complexes in H. pylori: (i) an affinity chromatography strategy based on aptamer-tagged sRNAs of interest to identify their protein binding partners; and (ii) a rapid method for chromosomal FLAG-tagging of proteins to facilitate co-immunoprecipitation of associated RNA species. Using these methods, we have identified RNA-protein interactions between the ribosomal protein S1 and various mRNAs and sRNAs of H. pylori. Moreover, both methods reported a stable RNA-protein complex between the abundant HPnc6910 sRNA and HP1334, a protein of unknown function that is encoded downstream of HPnc6910. Given that 50% of all bacteria may lack Hfq, our methods can be useful to identify RNA-protein interactions in a wider range of bacterial pathogens.

One of the most versatile riboswitch classes refers to purine nucleoside metabolism. In the cell, purine riboswitches of the respective mRNAs either act at the transcriptional or translational level and off- or on-regulate genes upon binding to their dedicated ligands. Biophysical studies on ligand-induced folding of these RNA domains in vitro contribute to understanding their regulation mechanisms in vivo. For such studies, in particular, for approaches using fluorescence spectroscopy, the preparation of large RNAs with site-specific chemical modifications is required. Here, we describe a strategy for the preparation of riboswitch aptamers and aptamers adjoined to their expression platforms by chemical synthesis and enzymatic ligation. The modular design enables fast access to a large number of purine riboswitch derivatives with the modification of interest at any strand position. We exemplarily provide a detailed protocol for the preparation of adenosine deaminase (add) A-riboswitch variants with 2-aminopurine (AP) modifications at the 40-nmol scale.