Branch site bulge conformations in domain 6 determine functional sugar puckers in group II intron splicing.

Although group II intron ribozymes are intensively studied the question how structural dynamics affects splicing catalysis has remained elusive. We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine. Our study determined a functional sugar pucker equilibrium between the transesterification active C2'-endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3'-endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step. Our detailed NMR spectroscopic investigation identified magnesium (II) ions and the branching reaction as regulators of the equilibrium populations. The tuneable secondary structure/sugar pucker equilibrium supports a conformational selection mechanism to up- and downregulate catalytically active and inactive states of the branch site adenosine to orchestrate the multi-step splicing process. The conformational dynamics of group II intron domain 6 is also proposed to be a key aspect for the directionality selection in reversible splicing.

NMR resonance assignments for the GTP-binding RNA aptamer 9-12 in complex with GTP.

Ligand binding RNAs such as artificially created RNA-aptamers are structurally highly diverse. Therefore, they represent important model systems for investigating RNA-folding, RNA-dynamics and the molecular recognition of chemically very different ligands, ranging from small molecules to whole cells. High-resolution structures of RNA-aptamers in complex with their cognate ligands often reveal unexpected tertiary structure elements. Recent studies on different classes of aptamers binding the nucleotide triphosphate GTP as a ligand showed that these systems not only differ widely in binding affinity but also in their ligand binding modes and structural complexity. We initiated the NMR-based structure determination of the high-affinity binding GTP-aptamer 9-12 in order to gain further insights into the diversity of ligand binding modes and structural variability of those aptamers. Here, we report 1H, 13C and 15N resonance assignments for the GTP 9-12-aptamer bound to GTP as the prerequisite for the structure determination by solution NMR.

The structure of the SAM/SAH-binding riboswitch.

S-adenosylmethionine (SAM) is a central metabolite since it is used as a methyl group donor in many different biochemical reactions. Many bacteria control intracellular SAM concentrations using riboswitch-based mechanisms. A number of structurally different riboswitch families specifically bind to SAM and mainly regulate the transcription or the translation of SAM-biosynthetic enzymes. In addition, a highly specific riboswitch class recognizes S-adenosylhomocysteine (SAH)-the product of SAM-dependent methyl group transfer reactions-and regulates enzymes responsible for SAH hydrolysis. High-resolution structures are available for many of these riboswitch classes and illustrate how they discriminate between the two structurally similar ligands SAM and SAH. The so-called SAM/SAH riboswitch class binds both ligands with similar affinities and is structurally not yet characterized. Here, we present a high-resolution nuclear magnetic resonance structure of a member of the SAM/SAH-riboswitch class in complex with SAH. Ligand binding induces pseudoknot formation and sequestration of the ribosome binding site. Thus, the SAM/SAH-riboswitches are translational 'OFF'-switches. Our results establish a structural basis for the unusual bispecificity of this riboswitch class. In conjunction with genomic data our structure suggests that the SAM/SAH-riboswitches might be an evolutionary late invention and not a remnant of a primordial RNA-world as suggested for other riboswitches.

5-Oxyacetic Acid Modification Destabilizes Double Helical Stem Structures and Favors Anionic Watson-Crick like cmo5 U-G Base Pairs.

Watson-Crick like G-U mismatches with tautomeric Genol or Uenol bases can evade fidelity checkpoints and thereby contribute to translational errors. The 5-oxyacetic acid uridine (cmo5 U) modification is a base modification at the wobble position on tRNAs and is presumed to expand the decoding capability of tRNA at this position by forming Watson-Crick like cmo5 Uenol -G mismatches. A detailed investigation on the influence of the cmo5 U modification on structural and dynamic features of RNA was carried out by using solution NMR spectroscopy and UV melting curve analysis. The introduction of a stable isotope labeled variant of the cmo5 U modifier allowed the application of relaxation dispersion NMR to probe the potentially formed Watson-Crick like cmo5 Uenol -G base pair. Surprisingly, we find that at neutral pH, the modification promotes transient formation of anionic Watson-Crick like cmo5 U- -G, and not enolic base pairs. Our results suggest that recoding is mediated by an anionic Watson-Crick like species, as well as bring an interesting aspect of naturally occurring RNA modifications into focus-the fine tuning of nucleobase properties leading to modulation of the RNA structural landscape by adoption of alternative base pairing patterns.

NMR resonance assignments for the SAM/SAH-binding riboswitch RNA bound to S-adenosylhomocysteine.

Riboswitches are structured RNA elements in the 5'-untranslated regions of bacterial mRNAs that are able to control the transcription or translation of these mRNAs in response to the specific binding of small molecules such as certain metabolites. Riboswitches that bind with high specificity to either S-adenosylmethionine (SAM) or S-adenosylhomocysteine (SAH) are widespread in bacteria. Based on differences in secondary structure and sequence these riboswitches can be grouped into a number of distinct classes. X-ray structures for riboswitch RNAs in complex with SAM or SAH established a structural basis for understanding ligand recognition and discrimination in many of these riboswitch classes. One class of riboswitches-the so-called SAM/SAH riboswitch class-binds SAM and SAH with similar affinity. However, this class of riboswitches is structurally not yet characterized and the structural basis for its unusual bispecificity is not established. In order to understand the ligand recognition mode that enables this riboswitch to bind both SAM and SAH with similar affinities, we are currently determining its structure in complex with SAH using NMR spectroscopy. Here, we present the NMR resonance assignment of the SAM/SAH binding riboswitch (env9b) in complex with SAH as a prerequisite for a solution NMR-based high-resolution structure determination.

Studying sparsely populated conformational states in RNA combining chemical synthesis and solution NMR spectroscopy.

Using chemical synthesis and solution NMR spectroscopy, RNA structural ensembles including a major ground state and minor populated excited states can be studied at atomic resolution. In this work, atom-specific 13C labeled RNA building blocks - a 5-13C-uridine and a 2,8-13C2-adenosine building block - are used to introduce isolated 13C-1H-spin topologies into a target RNA to probe such structural ensembles via NMR spectroscopy. First, the 5-13C-uridine 2'-O-TBDMS-phosphoramidite building block was introduced into a 21 nucleotide (nt) tP5c stem construct of the tP5abc subdomain of the Tetrahymena group I ribozyme. Then, the 2,8-13C2-adenosine 2'-O-TBDMS-phosphoramidite building block was incorporated into a 9 kDa and a 15 kD construct derived from the epsilon (ε) RNA element of the duck Hepatitis B virus. The 2,8-13C2-adenosine resonances of the 9 kDa 28 nt sequence could be mapped to the full-length 53 nt construct. The isolated NMR active nuclei pairs were used to probe for low populated excited states (<10%) via 13C-Carr-Purcell-Meiboom-Gill (CPMG)-relaxation dispersion NMR spectroscopy. The 13C-CPMG relaxation dispersion experiment recapitulated a secondary structure switching event in the P5c hairpin of the group I intron construct previously revealed by 15N relaxation dispersion experiments. In the ε-HBV RNA an unfolding event occurring on the millisecond time scale was found in the upper stem in-line with earlier observations. This unpaired conformational state is presumed to be important for the binding of the epsilon reverse transcriptase (RT) enzyme. Thus, a full description of an RNA's folding landscape helps to obtain a deeper understanding of its function, as these high energy conformational states often represent functionally important intermediates involved in (un)folding or ribozyme catalysis.