Active site dynamics in the lead-dependent ribozyme

Conformational dynamics are an important property of ribozymes and other RNA molecules but there is currently only limited information on the relationship between dynamics and RNA function. A recent structural study of the lead-dependent ribozyme, known as the leadzyme, showed significant dynamics at the active site and indicated that a structural rearrangement is required for the reaction to proceed from the ground to the transition state. In this work, microsecond-to-millisecond dynamics of the leadzyme are probed by analysis of the power dependence of (13)C NMR relaxation times in the rotating frame (T(1)(rho)). These results revealed a wide range of conformational dynamics for various residues in the leadzyme. For residue A25 in the active site, the power dependence of T(1)(rho) yielded an exchange lifetime similar to that previously measured by line-shape analysis, and provides an important calibration of this T(1)(rho) methodology for probing the dynamics of macromolecules. Strong evidence was also found for a previously suggested dynamic network of hydrogen bonds stabilizing the GAAA tetraloop motif. Within the active site of the leadzyme, internal motions are observed on a wide variety of time scales, suggesting a complex landscape of accessible states, and potential correlations between observed motions and catalytic function are discussed. These results demonstrate that the power dependence of (13)C T(1)(rho) relaxation times provides a valuable method for probing dynamics in nucleic acids.     

Crystal structure of the leadzyme at 1.8 A resolution: metal ion binding and the implications for catalytic mechanism and allo site ion regulation

The leadzyme is a small ribozyme, derived from in vitro selection, which catalyzes site specific, Pb(2+)-dependent RNA cleavage. Pb(2+) is required for activity; Mg(2+) inhibits activity, while many divalent and trivalent ions enhance it. The leadzyme structure consists of an RNA duplex interrupted by a trinucleotide bulge. Here, crystal structures determined to 1.8 A resolution, both with Mg(2+) as the sole divalent counterion and with Mg(2+) and Sr(2+) (which mimics Pb(2+) with respect to binding but not catalysis), reveal the metal ion interactions with both the ground state and precatalytic conformations of the leadzyme. Mg(H(2)O)(6)(2+) ions bridge complementary strands of the duplex at multiple locations by binding tandem purines of one RNA strand in the major groove. At one site, Mg(H(2)O)(6)(2+) ligates the phosphodiester backbone of the trinucleotide bulge in the ground state conformation, but not in the precatalytic conformation, suggesting (a) Mg(2+) may inhibit leadzyme activity by stabilizing the ground state and (b) metal ions which displace Mg(2+) from this site may activate the leadzyme. Binding of Sr(2+) to the presumed catalytic Pb(2+) site in the precatalytic leadzyme induces local structural changes in a manner that would facilitate alignment of the catalytic ribose 2'-hydroxyl with the scissile bond for cleavage. These data support a model wherein binding of a catalytic ion to a precatalytic conformation of the leadzyme, in conjunction with the flexibility of the trinucleotide bulge, may facilitate structural rearrangements around the scissle phosphodiester bond favoring configurations that allow bond cleavage.     

Structure of Poly (U).poly (A).poly (U)

The molecular structure of poly (U).poly (A).poly (U) has been determined and refined using the continuous x-ray intensity data on layer lines in the diffraction pattern obtained from an oriented fiber of the RNA. The final R-value for the preferred structure is 0.24, far lower than that for the plausible alternatives. The polymer forms an 11-fold right-handed triple-helix of pitch 33.5A and each base triplet is stabilized by Crick-Watson-Hoogsteen hydrogen bonds. The ribose rings in the three strands have C3'-endo, C2'-endo and C2'-endo conformations, respectively. The helix derives additional stability through systematic interchain hydrogen bonds involving ribose hydroxyls and uracil bases. The relatively grooveless cylindrical shape of the triple-helix is consistent with the lack of lateral organization.     

Leadzyme formed in vivo interferes with tobacco mosaic virus infection in Nicotiana tabacum

We developed a new method for inhibiting tobacco mosaic virus infection in tobacco plants based on specific RNA hydrolysis induced by a leadzyme. We identified a leadzyme substrate target sequence in genomic tobacco mosaic virus RNA and designed a 16-mer oligoribonucleotide capable of forming a specific leadzyme motif with a five-nucleotide catalytic loop. The synthetic 16-mer RNA was applied with nontoxic, catalytic amount of lead to infected tobacco leaves. We observed inhibition of tobacco mosaic virus infection in tobacco leaves in vivo due to specific tobacco mosaic virus RNA cleavage effected by leadzyme. A significant reduction in tobacco mosaic virus accumulation was observed even when the leadzyme was applied up to 2 h after inoculation of leaves with tobacco mosaic virus. This process, called leadzyme interference, is determined by specific recognition and cleavage of the target site by the RNA catalytic strand in the presence of Pb(2+).     

NMR studies of fully modified locked nucleic acid (LNA) hybrids: solution structure of an LNA:RNA hybrid and characterization of an LNA:DNA hybrid

LNA is a bicyclic nucleic acid analogue that contains one or more 2'-O,4'-C methylene linkage(s), which effectively locks the furanose ring in a C3'-endo conformation. We report here the NMR solution structure of a nonamer LNA:RNA hybrid and a structural characterization of a nonamer LNA:DNA hybrid, where the LNA strands are composed entirely of LNA nucleotides. This is the first structural characterization of fully modified LNA oligonucleotides. The high-resolution structure reveals that the LNA:RNA hybrid adopts an almost canonical A-type duplex morphology. The helix axis is almost straight and the duplex geometry is regular. This shows that fully modified LNA oligomers can hybridize with complementary RNA and form duplexes within the Watson-Crick framework. The LNA:DNA hybrid structurally resembles an RNA:DNA hybrid as shown by determination of deoxyribose sugar puckers and analysis of NOESY NMR spectra.     

Effect of substrate RNA sequence on the cleavage reaction by a short ribozyme.

Leadzyme is a ribozyme that requires Pb2+. The catalytic sequence, CUGGGAGUCC, binds to an RNA substrate, GGACC downward arrowGAGCCAG, cleaving the RNA substrate at one site. We have investigated the effect of the substrate sequence on the cleavage activity of leadzyme using mutant substrates in order to structurally understand the RNA catalysis. The results showed that leadzyme acted as a catalyst for single site cleavage of a C5 deletion mutant substrate, GGAC downward arrowGAGCCAG, as well as the wild-type substrate. However, a mutant substrate GGACCGACCAG, which had G8 deleted from the wild-type substrate, was not cleaved. Kinetic studies by surface plasmon resonance indicated that the difference between active and inactive structures reflected the slow association and dissociation rate constants of complex formation induced by Pb2+rather than differences in complex stability. CD spectra showed that the active form of the substrate-leadzyme complex was rearranged by Pb2+binding. The G8 of the wild-type substrate, which was absent in the inactive complex, is not near the cleavage site. Thus, these results show that the active substrate-leadzyme complex has a Pb2+binding site at the junction between the unpaired region (asymmetric internal loop) and the stem region, which is distal to the cleavage site. Pb2+may play a role in rearranging the bases in the asymmetric internal loop to the correct position for catalysis.     

Optical spectroscopic study of the effects of a single deoxyribose substitution in a ribose backbone: implications in RNA-RNA interaction

The 2'-OH group in the ribose sugars of an RNA molecule plays an important role in guiding tertiary interactions that stabilize different RNA structural motifs. Deoxyribose, or 2'-OH by 2'-H, substitution in both the single-stranded and the duplex part of an RNA backbone has been routinely used to evaluate what role the 2'-OH plays in different tertiary interactions that guide an RNA-RNA contact. A deoxyribose substitution not only has the effect of removing a hydrogen bond donating group, but also introduces a sugar moiety with a preference for C2'-endo pucker in a backbone of predominantly C3'-endo sugars. This study evaluates the effects of a single deoxyribose substitution in both single-stranded and double-helical forms of RNA oligomers. A single-stranded, nonrepetitive 7-mer oligoribonucleotide (7-mer RNA) and four different variants having the same base sequence but with a single deoxyribose sugar at different positions in the strands have been studied by ultraviolet (UV) absorption, circular dichroism (CD), and Fourier transform infrared (FTIR) spectroscopy. Duplexes were formed by association with the complementary strand of the 7-mer RNA. The results show that both RNA and DNA single strands have preorganized conformations with spectral properties resembling those of A- and B-form helices, respectively, with RNA being more heterogeneous than its DNA counterpart. A single deoxyribose substitution perturbs the structure of the RNA backbone, with the effect being more pronounced in the single-stranded than in the duplex structure. The perturbation depends on the position of the 2'-H substitution in the strand.     

Hydrostatic and osmotic pressure study of the RNA hydration

The tertiary structure of nucleic acids results from an equilibrium between electrostatic interactions of phosphates, stacking interactions of bases, hydrogen bonds between polar atoms and water molecules. Water interactions with ribonucleic acid play a key role in its structure formation, stabilization and dynamics. We used high hydrostatic pressure and osmotic pressure to analyze changes in RNA hydration. We analyzed the lead catalyzed hydrolysis of tRNA^Phe from S. cerevisiae as well as hydrolytic activity of leadzyme. Pb(II) induced hydrolysis of the single phosphodiester bond in tRNA^Phe is accompanied by release of 98 water molecules, while other molecule, leadzyme releases 86.     

Crystal structure of a lead-dependent ribozyme revealing metal binding sites relevant to catalysis

The leadzyme is a small RNA motif that catalyzes a site-specific, Pb2+-dependent cleavage reaction. As such, it is an example of a metal-dependent RNA enzyme. Here we describe the X-ray crystallographic structure of the leadzyme, which reveals two independent molecules per asymmetric unit. Both molecules feature an internal loop in which a bulged purine base stack twists away from the helical stem. This kinks the backbone, rendering the phosphodiester bond susceptible to cleavage. The independent molecules have different conformations: one leadzyme copy coordinates Mg2+, whereas the other binds only Ba2+ or Pb2+. In the active site of the latter molecule, a single Ba2+ ion coordinates the 2'-OH nucleophile, and appears to mimic the binding of catalytic lead. These observations allow a bond cleavage reaction to be modeled, which reveals the minimal structural features necessary for catalysis by this small ribozyme.     

UTP-bound and Apo structures of a minimal RNA uridylyltransferase

3'-Uridylylation of RNA is emerging as a phylogenetically widespread phenomenon involved in processing events as diverse as uridine insertion/deletion RNA editing in mitochondria of trypanosomes and small nuclear RNA (snRNA) maturation in humans. This reaction is catalyzed by terminal uridylyltransferases (TUTases), which are template-independent RNA nucleotidyltransferases that specifically recognize UTP and belong to a large enzyme superfamily typified by DNA polymerase beta. Multiple TUTases, recently identified in trypanosomes, as well as a U6 snRNA-specific TUTase enzyme in humans, are highly divergent at the protein sequence level. However, they all possess conserved catalytic and UTP recognition domains, often accompanied by various auxiliary modules present at the termini or between conserved domains. Here we report identification, structural and biochemical analyses of a novel trypanosomal TUTase, TbTUT4, which represents a minimal catalytically active RNA uridylyltransferase. The TbTUT4 consists of only two domains that define the catalytic center at the bottom of the nucleoside triphosphate and RNA substrate binding cleft. The 2.0 Angstroms crystal structure reveals two significantly different conformations of this TUTase: one molecule is in a relatively open apo conformation, whereas the other displays a more compact TUTase-UTP complex. A single nucleoside triphosphate is bound in the active site by a complex network of interactions between amino acid residues, a magnesium ion and highly ordered water molecules with the UTP's base, ribose and phosphate moieties. The structure-guided mutagenesis and cross-linking studies define the amino acids essential for catalysis, uracil base recognition, ribose binding and phosphate coordination by uridylyltransferases. In addition, the cluster of positively charged residues involved in RNA binding is identified. We also report a 2.4 Angstroms crystal structure of TbTUT4 with the bound 2' deoxyribonucleoside, which provides the structural basis of the enzyme's preference toward ribonucleotides.     

Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester

We analyzed cleavage of precursor tRNAs with an LNA, 2′-OCH₃, 2′-H or 2′-F modification at the canonical (c₀) site by bacterial RNase P. We infer that the major function of the 2′-substituent at nt −1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c₀ site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3′ -endo ribose and without the H-bond donor function of the 2′-substituent. LNA and 2′-OCH₃ suppressed processing at the major aberrant m₋₁ site; instead, the m₊₁ (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c₀ and m₊₁ sites had different pH profiles, with a higher Mg²⁺ requirement for c₀ versus m₊₁ cleavage. The strong catalytic defect for LNA and 2′-OCH₃ supports a model where the extra methylene (LNA) or methyl group (2′-OCH₃) causes a steric interference with a nearby bound catalytic Mg²⁺ during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects.     

Role of the catalytic metal during polymerization by DNA polymerase lambda

The incorporation of dNMPs into DNA by polymerases involves a phosphoryl transfer reaction hypothesized to require two divalent metal ions. Here we investigate this hypothesis using as a model human DNA polymerase lambda (Pol lambda), an enzyme suggested to be activated in vivo by manganese. We report the crystal structures of four complexes of human Pol lambda. In a 1.9 A structure of Pol lambda containing a 3'-OH and the non-hydrolyzable analog dUpnpp, a non-catalytic Na+ ion occupies the site for metal A and the ribose of the primer-terminal nucleotide is found in a conformation that positions the acceptor 3'-OH out of line with the alpha-phosphate and the bridging oxygen of the pyrophosphate leaving group. Soaking this crystal in MnCl2 yielded a 2.0 A structure with Mn2+ occupying the site for metal A. In the presence of Mn2+, the conformation of the ribose is C3'-endo and the 3'-oxygen is in line with the leaving oxygen, at a distance from the phosphorus atom of the alpha-phosphate (3.69 A) consistent with and supporting a catalytic mechanism involving two divalent metal ions. Finally, soaking with MnCl2 converted a pre-catalytic Pol lambda/Na+ complex with unreacted dCTP in the active site into a product complex via catalysis in the crystal. These data provide pre- and post-transition state information and outline in a single crystal the pathway for the phosphoryl transfer reaction carried out by DNA polymerases.     

X-ray crystal structure of a dimethylene sulfone-bridged ribonucleotide dimer in a single-stranded state.

A crystal structure has been solved for an analog of the r(ApU) ribodinucleotide, r(Aso2U), where a bridging non-ionic dimethylene sulfone linker replaces the phosphodiester linking group found in natural RNA. Crystals of the single-stranded state of r(Aso2U) were obtained from water at 50 degrees C. In these crystals, one hydrogen bond is formed between bases from different strands and base stacking occurs in intermolecular 'homo-A' and 'homo-U' stacks. Similar to typical oligoribonucleotides, the ribose rings adopt N-type conformations and dihedral angles chi are in the anti range. The all-trans rotamer of the CH2-SO2-CH2-CH2 bridge was found, which leads to a large adenine-uracil distance. Qualitative analysis of a NOESY spectrum of the Aso2U part in r(Uso2Cso2Aso2U) dissolved in a dimethylsulfoxide-D2O mixture indicates that the conformation observed in the crystal is also populated in solution. Comparison with the structure of r(Gso2C), which has been crystallized in the Watson-Crick paired state, shows that a rotation around zeta by +112 degrees leads from the observed, single-stranded state to a conformation that is compatible with formation of a duplex. A concerted trans/gauche flip of alpha and gamma then yields the standard conformer of A-type RNA helices. From the observed structure of r(Gso2C) and other oligonucleotides it is anticipated that this flip will also revert the ribose pucker from C2'-exo to C3'-endo.     

Structural characterization of three RNA hexanucleotide loops from the internal ribosome entry site of polioviruses.

Structural characteristics of three RNA hairpins from the internal ribosome entry site of poliovirus mRNAs have been determined in solution by NMR. Complete proton, phosphorus and carbon resonance assignments were made for the three 16 nt hairpins. The loop sequences, 5'-AAUCCA , AAACCA and GAACCA, have been shown to be essential for viral mRNA translation. NOESY spectra for the three oligomers were very similar indicating a common three dimensional structure. Stems were A-type duplexes with C3'-endo sugar pucker. In the loops, sequential base stacking interactions were detected for all bases except between U8/A8 and C9, indicating a turn in the phosphodiester backbone at this point. Only one nucleotide, U8/A8, had a sugar pucker which deviated appreciably from C3'-endo. The final base in the loop, A11, exhibited an unusual gauche (-) gamma angle. An ensemble of 10 structures calculated for one hairpin using restrained molecular dynamics shows that the first three bases of the loop are turned so as to be exposed to the exterior of the molecule, while the remaining three bases are in an orientation approximating a continuation of the stem helix. Structure calculations and NMR relaxation measurements indicate that the loop apex is subject to considerable local dynamics.     

5S rRNA is a leadzyme. A molecular basis for lead toxicity

This paper reports that the D-loop sequence of cellular mammalian ribosomal 5S RNAs is a natural leadzyme that specifically binds and cleaves in trans other RNA molecules in the presence of lead. The D-loops of these 5S rRNAs are similar in sequence to the active site of the leadzyme derived from tRNA(Phe), which cleaves a single bond in cis. We have devised a 12 nt model substrate based on the leadzyme sequence cleaved in trans by a 12 nt RNA molecule containing of the D-loop sequence. The model reaction occurs only at the appropriate concentration of lead and enzyme/substrate stoichiometry. The native 5S rRNA carries the same cleavage activity, although with different optimal lead concentration and stoichiometry. On the other hand, the isolated D-loop does not serve as a substrate when incubated with an RNA molecule with the potential to base pair with it and form the same internal loop (the bubble) present in the leadzyme-substrate complex. We show that the leadzyme cuts C-G, but not G-G or U-G linkages. The 5S rRNA leadzyme appears to have the shortest asymmetric pentanucleotide purine-rich loop flanked by two short double stranded RNAs. The leadzyme activity of native 5S rRNA may be an important aspect of lead toxicity in living cells. Because the leadzyme motif has been found in natural RNA species, its activity can be expressed in vivo even at a very low lead concentrations, of lead leading to the inactivation of other cellular RNAs. This might be one of the ways in which lead poisoning manifests itself at the molecular level. Lead toxicity is based not only on its binding to calcium and zinc binding proteins (such as Zn-fingers) and random hydrolysis of nucleic acids, but also, and most importantly, on the induction of the hydrolytic properties of RNA (RNA catalysis).     

Metal binding and base ionization in the U6 RNA intramolecular stem-loop structure

U6 RNA is a key component of the catalytic core of the spliceosome. A metal ion essential for the first catalytic step of pre-mRNA splicing binds to the U80 Sp phosphate oxygen within the yeast U6 intramolecular stem-loop (ISL). Here we present the first structural data for U6 RNA, revealing the three-dimensional structure of the highly conserved U6 ISL. The ISL binds metal ion at the U80 site with the same stereo specificity as the intact spliceosome. The metal-binding site is adjacent to a readily protonated C.A wobble pair. Protonation of the C.A pair and metal binding are mutually antagonistic. These results support a ribozyme model for U6 RNA function and suggest a possible mechanism for the regulation of RNA splicing.     

Nuclear Overhauser effect studies of the conformations of MgATP bound to the active and secondary sites of muscle pyruvate kinase

MgATP binds both at the active site (site 1) and at a secondary site (site 2) on each monomer of muscle pyruvate kinase as previously found by binding studies and by X-ray analysis. Interproton distances on MgATP bound at each site have been measured by the time-dependent nuclear Overhauser effect in the absence and presence of phosphoenolpyruvate (P-enolpyruvate), which blocks ATP binding at site 1. Interproton distances at site 2 are consistent with a single conformation of bound ATP with a high antiglycosidic torsional angle (chi = 68 +/- 10 degrees) and a C3'-endo ribose pucker (delta = 90 +/- 10 degrees). Interproton distances at site 1, determined in the absence of P-enolpyruvate by assuming the averaging of distances at both sites, cannot be fit by a single adenine-ribose conformation but require the contribution of at least three low-energy structures: 62 +/- 10% low anti (chi = 30 degrees), C3'-endo; 20 +/- 8% high anti (chi = 55 degrees), O1'-endo; and 18 +/- 8% syn (chi = 217 degrees), C2'-endo. Although a different set of ATP conformations might also have fit the interproton distances, the mixture of conformations used also fits previously determined distances from Mn2+ to the protons of ATP bound at site 1 [Sloan, D. L., & Mildvan, A. S. (1976) J. Biol. Chem. 251, 2412] and is similar to the adenine-ribose portion of free Co(NH3)4ATP, which consists of 35% low anti, 51% high anti, and 14% syn [Rosevear, P. R., Bramson, H. N., O'Brian, C., Kaiser, E. T., & Mildvan, A. S. (1983) Biochemistry 22, 3439].(ABSTRACT TRUNCATED AT 250 WORDS)     

A unique,thermostable dimer linkage structure of RD114 retroviral RNA

Retroviruses package their genome as RNA dimers linked together primarily by base-pairing between palindromic stem–loop (psl) sequences at the 5′ end of genomic RNA. Retroviral RNA dimers usually melt in the range of 55°C–70°C. However, RNA dimers from virions of the feline endogenous gammaretrovirus RD114 were reported to melt only at 87°C. We here report that the high thermal stability of RD114 RNA dimers generated from in vitro synthesized RNA is an effect of multiple dimerization sites located in the 5′ region from the R region to sequences downstream from the splice donor (SD) site. By antisense oligonucleotide probing we were able to map at least five dimerization sites. Computational prediction revealed a possibility to form stems with autocomplementary loops for all of the mapped dimerization sites. Three of them were located upstream of the SD site. Mutant analysis supported a role of all five loop sequences in the formation and thermal stability of RNA dimers. Four of the five psls were also predicted in the RNA of two baboon endogenous retroviruses proposed to be ancestors of RD114. RNA fragments of the 5′ R region or prolonged further downstream could be efficiently dimerized in vitro. However, this was not the case for the 3′ R region linked to upstream U3 sequences, suggesting a specific mechanism of negative regulation of dimerization at the 3′ end of the genome, possibly explained by a long double-stranded RNA region at the U3-R border. Altogether, these data point to determinants of the high thermostability of the dimer linkage structure of the RD114 genome and reveal differences from other retroviruses.     

Structural roles of monovalent cations in the HDV ribozyme

The hepatitis delta virus (HDV) ribozyme catalyzes viral RNA self-cleavage through general acid-base chemistry in which an active-site cytidine and at least one metal ion are involved. Monovalent metal ions support slow catalysis and were proposed to substitute for structural, but not catalytic, divalent metal ions in the RNA. To investigate the role of monovalent cations in ribozyme structure and function, we determined the crystal structure of the precursor HDV ribozyme in the presence of thallium ions (Tl(+)). Two Tl(+) ions can occupy a previously observed divalent metal ion hexahydrate-binding site located near the scissile phosphate, but are easily competed away by cobalt hexammine, a magnesium hexahydrate mimic and potent reaction inhibitor. Intriguingly, a third Tl(+) ion forms direct inner-sphere contacts with the ribose 2'-OH nucleophile and the pro-S(p) scissile phosphate oxygen. We discuss possible structural and catalytic implications of monovalent cation binding for the HDV ribozyme mechanism.