A glimpse into the active site of a group II intron and maybe the spliceosome, too.

The X-ray crystal structure of an excised group II self-splicing intron was recently solved by the Pyle group. Here we review some of the notable features of this structure and what they may tell us about the catalytic active site of the group II ribozyme and potentially the spliceosome. The new structure validates the central role of domain V in both the structure and catalytic function of the ribozyme and resolves several outstanding puzzles raised by previous biochemical, genetic and structural studies. While lacking both exons as well as the cleavage sites and nucleophiles, the structure reveals how a network of tertiary interactions can position two divalent metal ions in a configuration that is ideal for catalysis.

Internal bulge and tetraloop of the catalytic domain 5 of a group II intron ribozyme are flexible: implications for catalysis.

RNA molecules have an inherent flexibility that enables recognition of other interacting partners through potential disorder-order transitions, yet studies to quantify such motional dynamics remain few. With an increasing database of three-dimensional structures of biologically important RNA molecules, quantifying such motions becomes important to link structural deformations with function. One such system studied intensely is domain 5 (D5) from the self-splicing group II introns, which is at the heart of its catalytic machinery. We report the dynamics of a 36 nucleotide D5 from the Pylaiella littoralis group II intron in the presence and absence of magnesium ions, and at a range of temperatures (298K-318 K). Using high-resolution NMR experiments of heteronuclear nuclear Overhauser enhancement (NOE), spin-lattice (R(1)), and spin-spin (R(2)) (13)C relaxation rates, we determined the rotational diffusion tensor of D5 using the ROTDIF program modified for RNA dynamic analysis (ROTDIF_RNA). The D5 rotational diffusion tensor has an axial symmetric ratio (D(||)/D(perpendicular)) of 1.7+/-0.3, consistent with an estimated overall rotational correlation time of tau(m)=(2D(||)+4D(perpendicular))(-1) of 6.1(+/-0.3) ns at 298 K and 4.1(+/-0.2) ns at 318 K. The measured relaxation data were analyzed with the reduced spectral density mapping formalism using assumed values of the chemical shift anisotropy of the (13)C spins. Both the relaxation data and the values of the spectral density function reveal that the functional groups in D5 implicated in magnesium ion binding and catalysis (catalytic triad, internal bulge, and tetraloop regions) exhibit thermally induced motion on a wide variety of timescales. Because these motions parallel those observed in the intramolecular stem-loop of the U6 element within the spliceosome, we hypothesize that such extensive dynamic disorder likely facilitates D5 engaging both binding and catalytic regions of the ribozyme, and these may be a conserved feature of the catalytic machinery essential for catalysis.

Key labeling technologies to tackle sizeable problems in RNA structural biology.

The ability to adopt complex three-dimensional (3D) structures that can rapidly interconvert between multiple functional states (folding and dynamics) is vital for the proper functioning of RNAs. Consequently, RNA structure and dynamics necessarily determine their biological function. In the post-genomic era, it is clear that RNAs comprise a larger proportion (>50%) of the transcribed genome compared to proteins (< or =2%). Yet the determination of the 3D structures of RNAs lags considerably behind those of proteins and to date there are even fewer investigations of dynamics in RNAs compared to proteins. Site specific incorporation of various structural and dynamic probes into nucleic acids would likely transform RNA structural biology. Therefore, various methods for introducing probes for structural, functional, and biotechnological applications are critically assessed here. These probes include stable isotopes such as (2)H, (13)C, (15)N, and (19)F. Incorporation of these probes using improved RNA ligation strategies promises to change the landscape of structural biology of supramacromolecules probed by biophysical tools such as nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and Raman spectroscopy. Finally, some of the structural and dynamic problems that can be addressed using these technological advances are outlined.

Dynamic flexibility of double-stranded RNA activated PKR in solution.

PKR, an interferon-induced double-stranded RNA activated serine-threonine kinase, is a component of signal transduction pathways mediating cell growth control and responses to stress and viral infection. Analysis of separate PKR functional domains by NMR and X-ray crystallography has revealed details of PKR RNA binding domains and kinase domain, respectively. Here, we report the structural characteristics, calculated from biochemical and neutron scattering data, of a native PKR fraction with a high level of autophosphorylation and constitutive kinase activity. The experiments reveal association of the protein monomer into dimers and tetramers, in the absence of double-stranded RNA or other activators. Low-resolution structures of the association states were obtained from the large angle neutron scattering data and reveal the relative orientation of all protein domains in the activated kinase dimer. Low-resolution structures were also obtained for a PKR tetramer-monoclonal antibody complex. Taken together, this information leads to a new model for the structure of the functioning unit of the enzyme, highlights the flexibility of PKR and sheds light on the mechanism of PKR activation. The results of this study emphasize the usefulness of low-resolution structural studies in solution on large flexible multiple domain proteins.

Base flexibility in HIV-2 TAR RNA mapped by solution (15)N, (13)C NMR relaxation.

Binding of the HIV tat protein to the TAR (transactivating response region) RNA element activates transcription of the HIV viral genome. The complex of TAR with argininamide serves as a model for the RNA conformation in the tat-TAR complex. The dynamics of the HIV-2 TAR-argininamide complex was investigated by measurements of the relaxation rates of protonated base carbon and nitrogen nuclei. Six auto-correlation rates as well as cross-correlation rates were measured to map the frequencies of base motion in the HIV-2 TAR-argininamide complex. These measurements reveal a broad range of dynamic heterogeneity exhibited by hexanucleotide loop, the dinucleotide bulge, and the A-form helical regions. U23 in the bulge undergoes the largest dynamic change on binding argininamide, while U25 remains flexible, reflecting the large conformational change that is triggered upon ligand binding.

Fluorescence and solution NMR study of the active site of a 160-kDa group II intron ribozyme.

We have reconstructed the group II intron from Pylaiella littoralis (PL) into a hydrolytic ribozyme, comprising domains 1-3 (D123) connected in cis plus domain 5 (D5) supplied in trans that efficiently cleaves spliced exon substrates. Using a novel gel-based fluorescence assay and nuclear magnetic resonance (NMR) spectroscopy, we monitored the direct binding of D5 to D123, characterized the kinetics of the spliced exon hydrolysis reaction (which is mechanistically analogous to the reverse of the second catalytic step of splicing), and identified the binding surface of D123 on D5. This PL ribozyme acts as an RNA endonuclease even at low monovalent (100 mM KCl) and divalent ion concentrations (1-10 mM MgCl(2)). This is in contrast to other group II intron ribozyme systems that require high levels of salt, making NMR analysis problematic. D5 binds tightly to D123 with a K(d) of 650 +/- 250 nM, a K(m) of approximately 300 nM, and a K(cat) of 0.02 min(-1) under single turnover conditions. Within the approximately 160-kDa D123-D5 binary complex, site-specific binding to D123 leads to dramatic chemical shift perturbation of residues localized to the tetraloop and internal bulge within D5, suggesting a structural switch model for D5-assisted splicing. This minimal ribozyme thus recapitulates the essential features of the reverse of the second catalytic step and represents a well-behaved system for ongoing high-resolution structural work to complement folding and catalytic functional studies.

Structure of a self-splicing group II intron catalytic effector domain 5: parallels with spliceosomal U6 RNA.

Domain 5 (D5) is absolutely required for all catalytic functions of group II introns. Here we describe the solution NMR structure, electrostatic calculations, and detailed magnesium ion-binding surface of D5 RNA from the Pylaiella littoralis large ribosomal RNA intron (D5-PL). The overall structure consists of a hairpin capped by a GNRA tetraloop. The stem is divided into lower and upper helices of 8 and 5 bp, respectively, separated by an internal bulge. The D5-PL internal bulge nucleotides stack into the helical junction, resulting in a coupling between the bulge A25 and the closing base pair (G8-C27) of the lower helix. Comparison of the D5-PL structure to previously reported related structures indicates that our structure is most similar, in the helical regions, to the crystal structure of D5 from yeast Ai5gamma (D5-Ai5gamma) and the NMR structure of the U6 snRNA stem-loop region. Our structure differs in many respects from both the NMR and X-ray structures of D5-Ai5gamma in the bulge region. Electrostatic calculations and NMR chemical shift perturbation analyses reveal magnesium ion-binding sites in the tetraloop, internal bulge, and the AGC triad in the lower stem. Our results suggest that the structure, electrostatic environment, and the magnesium ion-binding sites within the tetraloop, bulge, and triad regions are conserved features of the splicing machinery of both the group II introns and the spliceosome that are likely key for catalytic function.

Resolution enhanced homonuclear carbon decoupled triple resonance experiments for unambiguous RNA structural characterization.

Large RNAs (>30 nucleotides) suffer from extensive resonance overlap that can seriously hamper unambiguous structural characterization. Here we present a set of 3D multinuclear NMR experiments with improved and optimized resolution and sensitivity for aiding with the assignment of RNA molecules. In all these experiments strong base and ribose carbon-carbon couplings are eliminated by homonuclear band-selective decoupling, leading to improved signal to noise and resolution of the C5, C6, and C1' carbon resonances. This decoupling scheme is applied to base-type selective 13C-edited NOESY, 13C-edited TOCSY (HCCH, CCH), HCCNH, and ribose H1C1C2 experiments. The 3D implementation of the HCCNH experiment with both carbon and nitrogen evolution enables direct correlation of 13C and 15N resonances at different proton resonant frequencies. The advantages of the new experiments are demonstrated on a 36 nucleotides hairpin RNA from domain 5 (D5) of the group II intron Pylaiella littoralis using an abbreviated assignment strategy. These four experiments provided additional separation for regions of the RNA that have overlapped chemical shift resonances, and enabled the assignment of critical D5 bulge nucleotides that could not be assigned using current experimental schemes.

Direct measurement of the 15N CSA/dipolar relaxation interference from coupled HSQC spectra.

Here we propose a method for the measurement of the (15)N CSA/dipolar relaxation interference based on direct comparison of the (15)N doublet components observed in a (1)H-coupled (1)H-(15)N HSQC-type spectrum. This allows the determination of the cross-correlation rates with no need for correction factors associated with other methods. The signal overlap problem of coupled HSQC spectra is addressed here by using the IPAP scheme (Ottiger et al., 1998). The approach is applied to the B3 domain of protein G to show that the method provides accurate measurements of the (15)N CSA/dipolar cross-correlation rates.