Towards understanding the catalytic core structure of the spliceosome.

The spliceosome catalyses the splicing of nuclear pre-mRNA (precursor mRNA) in eukaryotes. Pre-mRNA splicing is essential to remove internal non-coding regions of pre-mRNA (introns) and to join the remaining segments (exons) into mRNA before translation. The spliceosome is a complex assembly of five RNAs (U1, U2, U4, U5 and U6) and many dozens of associated proteins. Although a high-resolution structure of the spliceosome is not yet available, inroads have been made towards understanding its structure and function. There is growing evidence suggesting that U2 and U6 RNAs, of the five, may contribute to the catalysis of pre-mRNA splicing. In this review, recent progress towards understanding the structure and function of U2 and U6 RNAs is summarized.

A novel family of RNA tetraloop structure forms the recognition site for Saccharomyces cerevisiae RNase III.

RNases III are a family of double-stranded RNA (dsRNA) endoribonucleases involved in the processing and decay of a large number of cellular RNAs as well as in RNA interference. The dsRNA substrates of Saccharomyces cerevisiae RNase III (Rnt1p) are capped by tetraloops with the consensus sequence AGNN, which act as the primary docking site for the RNase. We have solved the solution structures of two RNA hairpins capped by AGNN tetraloops, AGAA and AGUU, using NMR spectroscopy. Both tetraloops have the same overall structure, in which the backbone turn occurs on the 3' side of the syn G residue in the loop, with the first A and G in a 5' stack and the last two residues in a 3' stack. A non-bridging phosphate oxygen and the universal G which are essential for Rnt1p binding are strongly exposed. The compared biochemical and structural analysis of various tetraloop sequences defines a novel family of RNA tetraloop fold with the consensus (U/A)GNN and implicates this conserved structure as the primary determinant for specific recognition of Rnt1p substrates.

Quantitative analysis of the isolated GAAA tetraloop/receptor interaction in solution: a site-directed spin labeling study.

The GNRA (N: any nucleotide; R: purine) tetraloop/receptor interaction is believed to be one of the most frequently occurring tertiary interaction motifs in RNAs, but an isolated tetraloop/receptor complex has not been identified in solution. In the present work, site-directed spin labeling is applied to detect tetraloop/receptor complex formation and estimate the free energy of interaction. For this purpose, the GAAA tetraloop/receptor interaction was chosen as a model system. A method was developed to place nitroxide labels at specific backbone locations in an RNA hairpin containing the GAAA tetraloop. Formation of the tetraloop/receptor complex was monitored through changes in the rotational correlation time of the tetraloop and the attached nitroxide. Results show that a hairpin containing the GAAA tetraloop forms a complex with an RNA containing the 11-nucleotide GAAA tetraloop receptor motif with an apparent Kd that is strongly dependent on Mg2+. At 125 mM MgCl2, Kd = 0.40 +/- 0.05 mM. The corresponding standard free energy of complex formation is -4.6 kcal/mol, representing the energetics of the tetraloop/receptor interaction in the absence of other tertiary constraints. The experimental strategy presented here should have broad utility in quantifying weak interactions that would otherwise be undetectable, for both nucleic acids and nucleic acid-protein complexes.

Structure and function of the small ribozymes.

Recently, major advances have been made toward increasing our understanding of small ribozyme structure and function. The first general acid-base catalytic mechanism for a ribozyme has been defined. Shifted nucleotide pK(a) values have been found to be surprisingly frequent structural elements. Finally, the dynamic nature of RNA catalysis has been highlighted through new structural and biochemical information.

Adenine protonation in domain B of the hairpin ribozyme.

Protein enzymes often use ionizable side chains, such as histidine, for general acid-base catalysis because the imidazole pK(a) is near neutral pH. RNA enzymes, on the other hand, are comprised of nucleotides which do not have apparent pK(a) values near neutral pH. Nevertheless, it has been recently shown that cytidine and adenine protonation can play an important role in both nucleic acid structure and catalysis. We have employed heteronuclear NMR methods to determine the pK(a) values and time scales of chemical exchanges associated with adenine protonation within the catalytically essential B domain of the hairpin ribozyme. The large, adenine-rich internal loop of the B domain allows us to determine adenine pK(a) values for a variety of non-Watson-Crick base pairs. We find that adenines within the internal loop have pK(a) values ranging from 4.8 to 5.8, significantly higher than the free mononucleotide pK(a) of 3. 5. Adenine protonation results in potential charge stabilization, hydrogen bond formation, and stacking interactions that are expected to stabilize the internal loop structure at low pH. Fast proton exchange times of 10-50 micros were determined for the well-resolved adenines. These results suggest that shifted pK(a) values may be a common feature of adenines in non-Watson-Crick base pairs, and identify two adenines which may participate in hairpin ribozyme active site chemistry.

Determination of metal ion binding sites within the hairpin ribozyme domains by NMR.

Cations play an important role in RNA folding and stabilization. The hairpin ribozyme is a small catalytic RNA consisting of two domains, A and B, which interact in the transition state in an ion-dependent fashion. Here we describe the interaction of mono-, di-, and trivalent cations with the domains of the ribozyme, as studied by homo- and heteronuclear NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping, and intermolecular NOEs indicate that the B domain contains four to five metal binding sites, which bind Mn(2+), Mg(2+), and Co(NH(3))(6)(3+). There is no significant structural change in the B domain upon the addition of Co(NH(3))(6)(3+) or Mg(2+). No specific monovalent ion binding sites exist on the B domain, as determined by (15)NH(4)(+) binding studies. In contrast to the B domain, there are no observable metal ion interactions within the internal loop of the A domain. Model structure calculations of Mn(2+) interactions at two sites within the B domain indicate that the binding sites comprise major groove pockets lined with functional groups oriented so that multiple hydrogen bonds can be formed between the RNA and Mn(H(2)O)(6)(2+) or Co(NH(3))(6)(3+). Site 1 is very similar in geometry to a site within the P4-P6 domain of the Tetrahymena group I intron, while site 2 is unique among known ion binding sites. The site 2 ion interacts with a catalytically essential nucleotide and bridges two phosphates. Due to its location and geometry, this ion may play an important role in the docking of the A and B domains.

Solution structure of the loop B domain from the hairpin ribozyme.

The hairpin ribozyme is a small catalytic RNA with a unique two-domain structure. Here we present the solution structure of the loop B domain of the hairpin ribozyme, which contains most of the catalytically essential nucleotides. The 38-nucleotide domain contains a 16-nucleotide internal loop that forms one of the largest non-Watson-Crick segments of base pairing thus far determined by either NMR or crystallography. Since the solution structure of the smaller loop A domain has been previously solved, an NMR structure-based model of the 22,000 Mr hairpin ribozyme-substrate open complex emerges by joining the two domain structures. Strikingly, catalytically essential functional groups for the loop B domain are concentrated within an expanded minor groove, presenting a clear docking surface for the loop A domain.

Mutant ATP-binding RNA aptamers reveal the structural basis for ligand binding.

The solution structure of the ATP-binding RNA aptamer has recently been determined by NMR spectroscopy. The three-dimensional fold of the molecule is determined to a large extent by stacking and hydrogen bond interactions. In the course of the structure determination it was discovered that several highly conserved nucleotides in the binding pocket can be substituted while retaining binding under NMR conditions. These surprising findings allow a closer look at the interactions that determine stability and specificity of the aptamer as well as local structural features of the molecule. The binding properties of ATP binder mutants and modified ligand molecules are explored using NMR spectroscopy, column binding studies and molecular modeling. We present additional evidence and new insights regarding the network of hydrogen bonds that defines the structure and determines stability and specificity of the aptamer.

Solution structure of the conserved 16 S-like ribosomal RNA UGAA tetraloop.

The solution structure of the highly conserved UGAA tetraloop found at the 3' end of eukaryotic 16 S-like ribosomal RNA has been solved by nuclear magnetic resonance spectroscopy in the form of the 12 nucleotide hairpin 5'-GGUG[UGAA]CACC. The UGAA tetraloop displays a novel fold. The backbone turn occurs between the G and the third A in the loop, with the U and G in a 5' stack and the As in a 3' stacking arrangement. The loop is closed by a U-A mismatch in which the O2, 2'OH, and O4' groups of the U are within hydrogen bonding distance of the amino group of the A. The tetraloop does not make a uridine-turn, even though its sequence is identical to a U-turn found within the anticodon loop of tRNA(Phe). The hydrogen bonding pattern in the tetraloop provides insight into the function of base modifications found in vivo within this portion of 16 S-like rRNA.

Solution structure of a GAAA tetraloop receptor RNA.

The GAAA tetraloop receptor is an 11-nucleotide RNA sequence that participates in the tertiary folding of a variety of large catalytic RNAs by providing a specific binding site for GAAA tetraloops. Here we report the solution structure of the isolated tetraloop receptor as solved by multidimensional, heteronuclear magnetic resonance spectroscopy. The internal loop of the tetraloop receptor has three adenosines stacked in a cross-strand or zipper-like fashion. This arrangement produces a high degree of base stacking within the asymmetric internal loop without extrahelical bases or kinking the helix. Additional interactions within the internal loop include a U. U mismatch pair and a G.U wobble pair. A comparison with the crystal structure of the receptor RNA bound to its tetraloop shows that a conformational change has to occur upon tetraloop binding, which is in good agreement with previous biochemical data. A model for an alternative binding site within the receptor is proposed based on the NMR structure, phylogenetic data and previous crystallographic structures of tetraloop interactions.

Through-bond correlation of imino and aromatic resonances in 13C-, 15N-labeled RNA via heteronuclear TOCSY.

Novel HCCNH TOCSY NMR experiments are presented that provide unambiguous assignment of the exchangeable imino proton resonances by intranucleotide through-bond connectivities to the (assigned) nonexchangeable purine H8 and pyrimidine H6 protons in uniformly 15N-, 13C-labeled RNA oligonucleotides. The HCCNH TOCSY experiments can be arranged as a two-dimensional experiment, correlating solely GH8/UH6 and GH1/UH3 proton resonances (HCCNH), 51 as three-dimensional experiments, in which additional chemical shift labeling either by GN1/UN3 (HCCNH) or by GC8/UC6 (HCCNH) chemical shifts is introduced. The utility of these experiments for the assignment of relatively large RNA oligonucleotides is demonstrated for two different RNA molecules.

Reconstitution of hairpin ribozyme activity following separation of functional domains.

The hairpin ribozyme is a 50-nucleotide RNA enzyme of unknown three-dimensional structure. Here, we, demonstrate that interdomain interactions are required for catalytic function by reconstitution of activity following separation of an essential, independently folding domain (loop B) from the substrate binding strand at a helical junction. The resulting construct relies on long range tertiary contacts for catalysis. For this work, we used an optimized ribozyme and substrate, which included sequence changes to minimize the formation of nonproductive conformational isomers. Kinetic analysis was carried out using both single and multiple turnover methods and shows that the catalytic efficiency (kcat/Km) of the reconstituted ribozyme is 10(4)-fold lower than that of the intact ribozyme. The decrease in kcat/Km results entirely from a 10(4)-fold increase in the apparent Km, whereas the kcat parameter is essentially unchanged. Therefore, cleavage chemistry appears to be unimpaired, but the reaction is limited by the productive assembly of the two domains. Our results strongly support a previously proposed model in which the catalytic topology of the ribozyme contains a bend at a helical junction.

Structure-mapping of the hairpin ribozyme. Magnesium-dependent folding and evidence for tertiary interactions within the ribozyme-substrate complex.

We have used chemical modification analysis to probe the solution structure of the hairpin ribozyme. The modifying reagents dimethylsulfate, 1-cyclohexyl-N'-[2-(N-methylmorpholino) ethyl-carbodiimide-p-toluenesulfonate, kethoxal, diethylpyrocarbonate and (2,12-dimethyl-3,7,11,17- tetraazabicyclo [11.3.1]heptadeca-1(17),2,11,13,15-pentaenato) nickel(II) perchlorate were used to probe functional groups that participate in Watson-Crick and non-canonical base-pairs. Our results confirm the existence of four short helices (3 to 6 bp) within the ribozyme-substrate complex, and demonstrate that one intramolecular helix (helix 4) is comprised of three base-pairs rather than the previously suggested five. In the absence of magnesium, the ribozyme is observed to fold into its secondary structure. Upon addition of magnesium, a striking difference in chemical modification is observed, particularly at sites within the ribozyme's large internal loop (loop B) that are essential for catalytic function (bases 21 to 26). Moreover, magnesium-dependent folding clearly destabilizes an A-U base-pair in a region where a proposed bend is required to juxtapose the catalytically essential loops A and B. Upon addition of substrate, no changes are observed in the structure of helix 3, loop B or helix 4. However, strong protection of bases in the substrate-binding domain is observed, including those located across internal loop A. The modification data are consistent with the formation of a previously proposed tertiary structure motif within loop B that includes non-canonical G-A, A-U and A-A base-pairs, and that is identical with those identified by NMR analysis of loop E of 5 S rRNA and the sarcin/ricin loop of 28 S rRNA. Our results indicate that the hairpin ribozyme adopts a stable magnesium-dependent tertiary structure to which the substrate binds without inducing major conformational changes, and that substrate recognition is likely to involve non-canonical base-pairs between the ribozyme and substrate sequences adjacent to the cleavage site.

A photo-cross-linkable tertiary structure motif found in functionally distinct RNA molecules is essential for catalytic function of the hairpin ribozyme.

We have identified an essential UV-sensitive tertiary structure domain within the hairpin ribozyme. Irradiation at 254 nm produces two cross-linked RNA species that are resolved from the unmodified structure by denaturing gel electrophoresis. One cross-link forms at high efficiency and maps between nucleotides G21 and/or A22 and U41, all essential bases located within an internal loop joining helices 3 and 4. A second cross-link forms between nucleotides A20 and U42 as a result of ribozyme dimerization at concentrations greater than 0.5 microM. Both cross-linked species retain cleavage activity and so presumably reflect catalytically proficient structures of the ribozyme. Formation of the intramolecular cross-link is independent of Mg2+ and substrate and is blocked by base substitutions within the reactive domain that inhibit catalysis. A 36-nt RNA fragment containing the photoreactive domain but lacking the substrate binding domain also cross-links with high efficiency and maps between G21 and U41, as observed with the intact molecule. The sequence and cross-linking sites of the UV-sensitive internal loop are strikingly similar to those found in several other RNA molecules, including loop E of 5S rRNA. These results suggest that the loop E-like structure may be a common RNA folding domain that is utilized in a variety of functionally important RNA molecules.

Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme.

In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived.

Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates.

Substrate recognition by the hairpin ribozyme has been proposed to involve two short intermolecular helices, termed helix 1 and helix 2. We have used a combination of three methods (cleavage of mismatched substrates, in vitro selection, and site-specific mutational analysis) to systematically determine the substrate recognition rules for this RNA enzyme. Assays measuring substrate cleavage in trans under multiple turnover conditions were conducted using the wild-type ribozyme and substrates containing mismatches in all sites potentially recognized by the ribozyme. Molecules containing single- and multiple-base mismatches in helix 2 at sites distant from the cleavage site (g-4c, u-5a, g-4c: u-5a) were cleaved with reduced efficiency, whereas those with mismatches proximal to the cleavage site (c-2a, a-3c, c-2a: a-3c) were not cut. Analogous results were obtained for helix 1, where mismatches distal from the cleavage site (u+7a, u+8a, u+9a, u+7a: u+8a: u+9a) were used much more efficiently than those proximal to the cleavage site (c+4a, u-5a, g+6c, c+4a: u+5a: g+6c). In vitro selection experiments were carried out to identify active variants from populations of molecules in which either helix 1 or helix 2 was randomized. Results constitute an artificial phylogenetic data base that proves base-pairing of nucleotides at five positions within helix 1 and three positions within helix 2 and reveals a significant sequence bias at 3 bp (c+4.G6, c-2.G11, and a-3.U12). This sequence bias was confirmed at two sites by measuring relative cleavage rates of all 16 possible dinucleotide combinations at base pairs c+4.G6 and c-2.G11.(ABSTRACT TRUNCATED AT 250 WORDS)