Ronald Micura

Structural and functional insights into 5′-ppp RNA pattern recognition by the innate immune receptor RIG-I

RIG-I is a cytosolic helicase that senses 5′-ppp RNA contained in negative-strand RNA viruses and triggers innate antiviral immune responses. Calorimetric binding studies established that the RIG-I C-terminal regulatory domain (CTD) binds to blunt-end double-stranded 5′-ppp RNA a factor of 17 more tightly than to its single-stranded counterpart. Here we report on the crystal structure of RIG-I CTD bound to both blunt ends of a self-complementary 5′-ppp dsRNA 12-mer, with interactions involving 5′-pp clearly visible in the complex. The structure, supported by mutation studies, defines how a lysine-rich basic cleft within the RIG-I CTD sequesters the observable 5′-pp of the bound RNA, with a stacked phenylalanine capping the terminal base pair. Key intermolecular interactions observed in the crystalline state are retained in the complex of 5′-ppp dsRNA 24-mer and full-length RIG-I under in vivo conditions, as evaluated from the impact of binding pocket RIG-I mutations and 2′-OCH3 RNA modifications on the interferon response.

Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

The majority of structural efforts addressing RNAs catalytic function have focused on natural ribozymes, which catalyze phosphodiester transfer reactions. By contrast, little is known about how RNA catalyzes other types of chemical reactions. We report here the crystal structures of a ribozyme that catalyzes enantioselective carbon-carbon bond formation by the Diels-Alder reaction in the unbound state and in complex with a reaction product. The RNA adopts a λ-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, complementarity and electronic effects. We observe structural parallels in the independently evolved catalytic pocket architectures for ribozyme- and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions.

Ligand-induced folding of the adenosine deaminase A-riboswitch and implications on riboswitch translational control.

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.

Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach

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 ligands 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.

Nascent Peptide in the Ribosome Exit Tunnel Affects Functional Properties of the A-Site of the Peptidyl Transferase Center

The ability to monitor the nascent peptide structure and to respond functionally to specific nascent peptide sequences is a fundamental property of the ribosome. An extreme manifestation of such response is nascent peptide-dependent ribosome stalling, involved in the regulation of gene expression. The molecular mechanisms of programmed translation arrest are unclear. By analyzing ribosome stalling at the regulatory cistron of the antibiotic resistance gene ermA, we uncovered a carefully orchestrated cooperation between the ribosomal exit tunnel and the A-site of the peptidyl transferase center (PTC) in halting translation. The presence of an inducing antibiotic and a specific nascent peptide in the exit tunnel abrogate the ability of the PTC to catalyze peptide bond formation with a particular subset of amino acids. The extent of the conferred A-site selectivity is modulated by the C-terminal segment of the nascent peptide, where the third-from-last residue plays a critical role.

Folding of a transcriptionally acting PreQ1 riboswitch

7-Aminomethyl-7-deazaguanine (preQ1) sensitive mRNA domains belong to the smallest riboswitches known to date. Although recent efforts have revealed the three-dimensional architecture of the ligand–aptamer complex less is known about the molecular details of the ligand-induced response mechanism that modulates gene expression. We present an in vitro investigation on the ligand-induced folding process of the preQ1 responsive RNA element from Fusobacterium nucleatum using biophysical methods, including fluorescence and NMR spectroscopy of site-specifically labeled riboswitch variants. We provide evidence that the full-length riboswitch domain adopts two different coexisting stem-loop structures in the expression platform. Upon addition of preQ1, the equilibrium of the competing hairpins is significantly shifted. This system therefore, represents a finely tunable antiterminator/terminator interplay that impacts the in vivo cellular response mechanism. A model is presented how a riboswitch that provides no obvious overlap between aptamer and terminator stem-loop solves this communication problem by involving bistable sequence determinants.

Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution

Thiamine pyrophosphate (TPP)-sensitive mRNA domains are the most prevalent riboswitches known. Despite intensive investigation, the complex ligand recognition and concomitant folding processes in the TPP riboswitch that culminate in the regulation of gene expression remain elusive. Here, we used single-molecule fluorescence resonance energy transfer imaging to probe the folding landscape of the TPP aptamer domain in the absence and presence of magnesium and TPP. To do so, distinct labeling patterns were used to sense the dynamics of the switch helix (P1) and the two sensor arms (P2/P3 and P4/P5) of the aptamer domain. The latter structural elements make interdomain tertiary contacts (L5/P3) that span a region immediately adjacent to the ligand-binding site. In each instance, conformational dynamics of the TPP riboswitch were influenced by ligand binding. The P1 switch helix, formed by the 5′ and 3′ ends of the aptamer domain, adopts a predominantly folded structure in the presence of Mg2+ alone. However, even at saturating concentrations of Mg2+ and TPP, the P1 helix, as well as distal regions surrounding the TPP-binding site, exhibit an unexpected degree of residual dynamics and disperse kinetic behaviors. Such plasticity results in a persistent exchange of the P3/P5 forearms between open and closed configurations that is likely to facilitate entry and exit of the TPP ligand. Correspondingly, we posit that such features of the TPP aptamer domain contribute directly to the mechanism of riboswitch-mediated translational regulation.

2'-Azido RNA, a versatile tool for chemical biology: synthesis, X-ray structure, siRNA applications, click labeling.

Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2′-azido cytidine and 2′-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2′-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2′-azido uridine and 2′-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2′-azido group supports the C3′-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2′-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2′-azido modifications are compatible with 2′-fluoro and/or 2′-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2′-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2′-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification.

Long non-coding RNAs as targets for cytosine methylation.

Post-synthetic modifications of nucleic acids have long been known to affect their functional and structural properties. For instance, numerous different chemical modifications modulate the structural organization, stability or translation efficiency of tRNAs and rRNAs. In contrast, little is known about modifications of poly(A)RNAs. Here, we demonstrate for the first time that the two well-studied regulatory long non-coding RNAs HOTAIR and XIST are targets of site-specific cytosine methylation. In both XIST and HOTAIR, we found methylated cytosines located within or near functionally important regions that are known to mediate interaction with chromatin-associated protein complexes. We show that cytosine methylation in the XIST A structure strongly affects binding to the chromatin-modifying complex PRC2 in vitro. These results suggest that cytosine methylation may serve as a general strategy to regulate the function of long non-coding RNAs.

Chemical engineering of the peptidyl transferase center reveals an important role of the 2′-hydroxyl group of A2451

The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystallography showed that A2451 of 23S rRNA makes the closest approach to the attacking amino group of aminoacyl-tRNA. Mutations of A2451 had relatively small effects on transpeptidation and failed to unequivocally identify the crucial functional group(s). Here, we employed an in vitro reconstitution system for chemical engineering the peptidyl transferase center by introducing non-natural nucleosides at position A2451. This allowed us to investigate the peptidyl transfer reaction performed by a ribosome that contained a modified nucleoside at the active site. The main finding is that ribosomes carrying a 2′-deoxyribose at A2451 showed a compromised peptidyl transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A2451 had only little impact on peptide bond formation, as long as the 2′-hydroxyl was present. This implicates a functional or structural role of the 2′-hydroxyl group at A2451 for transpeptidation.