Role of an active site adenine in hairpin ribozyme catalysis

The hairpin ribozyme is a small catalytic RNA that accelerates reversible cleavage of a phosphodiester bond. Structural and mechanistic studies suggest that divalent metals stabilize the functional structure but do not participate directly in catalysis. Instead, two active site nucleobases, G8 and A38, appear to participate in catalytic chemistry. The features of A38 that are important for active site structure and chemistry were investigated by comparing cleavage and ligation reactions of ribozyme variants with A38 modifications. An abasic substitution of A38 reduced cleavage and ligation activity by 14,000-fold and 370,000-fold, respectively, highlighting the critical role of this nucleobase in ribozyme function. Cleavage and ligation activity of unmodified ribozymes increased with increasing pH, evidence that deprotonation of some functional group with an apparent pK(a) value near 6 is important for activity. The pH-dependent transition in activity shifted by several pH units in the basic direction when A38 was substituted with an abasic residue, or with nucleobase analogs with very high or low pK(a) values that are expected to retain the same protonation state throughout the experimental pH range. Certain exogenous nucleobases that share the amidine group of adenine restored activity to abasic ribozyme variants that lack A38. The pH dependence of chemical rescue reactions also changed according to the intrinsic basicity of the rescuing nucleobase, providing further evidence that the protonation state of the N1 position of purine analogs is important for rescue activity. These results are consistent with models of the hairpin ribozyme catalytic mechanism in which interactions with A38 provide electrostatic stabilization to the transition state.     

锤头型核酶作用机理的研究进展

概述了锤头型核酶的二级结构特征,动力学反应的特点以及核酶切割反应的催化机制,提出了锤头型核酶作用机理有待于深入研究的问题.     

作用于HBV（adr亚型）RNA的tRNA—包埋锤头状核酶的研究

设计了针对ＨＢＶ（ａｄｒ亚型）ＲＮＡ的二个锤头状核酶（ＲＳ３和ＲＣ２）,并将其插入ｔＲＮＡ反密码环中（ＲｔＳ３和ＲｔＣ２）,以增加其稳定性。实验表明,虽然插入ｔＲＮＡ中的核酶与裸露核酶相比,催化活性有所下降,但在胎牛血清和ＨｅｐＧ２细胞抽提液中的稳定性却明显提高。因此,ｔＲＮＡ－包埋核酶有可能提高在体内的抗病毒能力。     

Structural metals in the group I intron: a ribozyme with a multiple metal ion core

Metal ions play key roles in the folding and function for many structured RNAs, including group I introns. We determined the X-ray crystal structure of the Azoarcus bacterial group I intron in complex with its 5' and 3' exons. In addition to 222 nucleotides of RNA, the model includes 18 Mg(2+) and K(+) ions. Five of the metals bind within 12 A of the scissile phosphate and coordinate the majority of the oxygen atoms biochemically implicated in conserved metal-RNA interactions. The metals are buried deep within the structure and form a multiple metal ion core that is critical to group I intron structure and function. Eight metal ions bind in other conserved regions of the intron structure, and the remaining five interact with peripheral structural elements. Each of the 18 metals mediates tertiary interactions, facilitates local bends in the sugar-phosphate backbone or binds in the major groove of helices. The group I intron has a rich history of biochemical efforts aimed to identify RNA-metal ion interactions. The structural data are correlated to the biochemical results to further understand the role of metal ions in group I intron structure and function.     

Terbium-mediated footprinting probes a catalytic conformational switch in the antigenomic hepatitis delta virus ribozyme

The two forms of the hepatitis delta virus ribozyme are derived from the genomic and antigenomic RNA strands of the human hepatitis delta virus (HDV), where they serve a crucial role in pathogen replication by catalyzing site-specific self-cleavage reactions. The HDV ribozyme requires divalent metal ions for formation of its tertiary structure, consisting of a tight double-nested pseudoknot, and for efficient self- (or cis-) cleavage. Comparison of recently solved crystal structures of the cleavage precursor and 3' product indicates that a significant conformational switch is required for catalysis by the genomic HDV ribozyme. Here, we have used the lanthanide metal ion terbium(III) to footprint the precursor and product solution structures of the cis-acting antigenomic HDV ribozyme. Inhibitory Tb(3+) binds with high affinity to similar sites on RNA as Mg(2+) and subsequently promotes slow backbone scission. We find subtle, yet significant differences in the terbium(III) footprinting pattern between the precursor and product forms of the antigenomic HDV ribozyme, consistent with differences in conformation as observed in the crystal structures of the genomic ribozyme. In addition, UV melting profiles provide evidence for a less tight tertiary structure in the precursor. In both the precursor and product we observe high-affinity terbium(III) binding sites in joining sequence J4/2 (Tb(1/2) approximately 4 microM) and loop L3, which are key structural components forming the catalytic core of the HDV ribozyme, as well as in several single-stranded regions such as J1/2 and the L4 tetraloop (Tb(1/2) approximately 50 microM). Sensitized luminescence spectroscopy confirms that there are at least two affinity classes of Tb(3+) binding sites. Our results thus demonstrate that a significant conformational change accompanies catalysis in the antigenomic HDV ribozyme in solution, similar to the catalytic conformational switch observed in crystals of the genomic form, and that structural and perhaps catalytic metal ions bind close to the catalytic core.     

Folding of the hammerhead ribozyme: pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme-substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair.     

Productive folding to the native state by a group II intron ribozyme.

Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions. Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding. Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5gamma. The isotherms span a similar range of Mg(2+) concentrations and share a similar index of cooperativity. Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far. The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error. Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5gamma intron is attributable to the presence of unproductive kinetic traps along the folding pathway. Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5gamma occurs early along the reaction pathway. We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure.     

A pH-dependent conformational change, rather than the chemical step, appears to be rate-limiting in the hammerhead ribozyme cleavage reaction.

We have investigated the chemical basis for a previously observed 7.8 A conformational change in the hammerhead ribozyme that positions the substrate for in-line attack. We have found that the conformational change can only be observed at or above pH 8.5 (in the presence of Co(2+)) and requires the presence of an ionizable 2'-OH at the cleavage site, and note that this observed apparent pK(a) of 8.5 for the conformational change is within experimental error (+/-0.5) of the previously reported apparent kinetic pK(a) of 8.5 for the hammerhead ribozyme in the presence of Co(2+). We have solved two crystal structures of hammerhead ribozymes having 2'-OCH(3) or 2'-F substitutions at the cleavage site and have found that these will not undergo a conformational change equivalent to that observed for the hammerhead ribozyme having an unmodified attacking nucleophile under otherwise identical conditions. We have also characterized the kinetics of cleavage in the crystal. In addition to verifying that the particular sequence of RNA that we crystallized cleaves faster in the crystal than in solution, we also find that the extent of cleavage in the crystal is complete, unlike in solution where this and most other hammerhead ribozyme substrates are cleaved only to about 70 % completion. The initial cleavage rate in the crystal obeys the expected log-linear relation between cleavage-rate and pH with a slope of 0.7, as has been observed for other hammerhead ribozyme sequences in solution, indicating that in both the crystal and in solution the pH-dependent step is rate-limiting. However, the cleavage rate in the crystal is biphasic, with the most dramatic distinction between initial (slower) and final (faster) phases appearing at pH 6.0. The initial phase corresponds to the pH-dependent cleavage rate observed in solution, but the second, faster phase is roughly pH-independent and closely parallels the cleavage rate observed at pH 8 (0.4/minute). This result is particularly remarkable because it entails that the rapidly cleaving phase at pH 6 is comparable to the cleavage rate for the fastest cleaving hammerhead ribozymes at pH 6. Based upon these observations, we conclude that the pH-dependent conformational change is the rate-determining step under standard conditions for the hammerhead ribozyme self-cleavage reaction, and that an ionizable 2'-proton at cleavage site is required for this conformational change. We further hypothesize that deprotonation of the cleavage-site 2'-oxygen drives this conformational change.     

RNA tertiary interactions mediate native collapse of a bacterial group I ribozyme

Large RNAs collapse into compact intermediates in the presence of counterions before folding to the native state. We previously found that collapse of a bacterial group I ribozyme correlates with the formation of helices within the ribozyme core, but occurs at Mg2+ concentrations too low to support stable tertiary structure and catalytic activity. Here, using small-angle X-ray scattering, we show that Mg2+-induced collapse is a cooperative folding transition that can be fit by a two-state model. The Mg2+ dependence of collapse is similar to the Mg2+ dependence of helix assembly measured by partial ribonuclease T1 digestion and of an unfolding transition measured by UV hypochromicity. The correspondence between multiple probes of RNA structure further supports a two-state model. A mutation that disrupts tertiary contacts between the L9 tetraloop and its helical receptor destabilized the compact state by 0.8 kcal/mol, while mutations in the central triplex were less destabilizing. These results show that native tertiary interactions stabilize the compact folding intermediates under conditions in which the RNA backbone remains accessible to solvent.     

针对不同基因靶位的锤头状核酶对HBV的抑制作用研究

前基因组mRNA是HBV(Hepatitis Bvirus)基因表达和复制的重要中间产物,全长的前基因组mRNA分子具有复杂易变的二级结构,是设计抑制HBV的核酶时所必须考虑的因素。我们使用多个最新的计算机软件对HBV前基因组mRNA二级结构进行模拟、分析,在全面分析核酶的可作用位点的基础上设计三个针对不同基因靶位的锤头状核酶,并对它们在细胞中对HBV的抑制作用进行研究。结果表明在HBV前基因组mRNA上存在几个高度复杂二级结构的区域,可能对核酶完全不敏感,而S、C、X基因的编码区是合适的核酶作用位点,都可达到对HBV的有效抑制,而且X基因位点的核酶对HBV的各种mRNA的抑制作用最为明显,是设计针对HBV核酶时应该优先考虑的位点。     

Conservation of helical structure contributes to functional metal ion interactions in the catalytic domain of ribonuclease P RNA

Like protein enzymes, catalytic RNAs contain conserved structure motifs important for function. A universal feature of the catalytic domain of ribonuclease P RNA is a bulged-helix motif within the P1-P4 helix junction. Here, we show that changes in bulged nucleotide identity and position within helix P4 affect both catalysis and substrate binding, while a subset of the mutations resulted only in catalytic defects. We find that the proximity of the bulge to sites of metal ion coordination in P4 is important for catalysis; moving the bulge distal to these sites and deleting it had similarly large effects, while moving it proximal to these sites had only a moderate effect on catalysis. To test whether the effects of the mutations are linked to metal ion interactions, we used terbium-dependent cleavage of the phosphate backbone to probe metal ion-binding sites in the wild-type and mutant ribozymes. We detect cleavages at specific sites within the catalytic domain, including helix P4 and J3/4, which have previously been shown to participate directly in metal ion interactions. Mutations introduced into P4 cause local changes in the terbium cleavage pattern due to alternate metal ion-binding configurations with the helix. In addition, a bulge deletion mutation results in a 100-fold decrease in the single turnover cleavage rate constant at saturating magnesium levels, and a reduced affinity for magnesium ions important for catalysis. In light of the alternate terbium cleavage pattern in P4 caused by bulge deletion, this decreased ability to utilize magnesium ions for catalysis appears to be due to localized structural changes in the ribozyme's catalytic core that weaken metal ion interactions in P4 and J3/4. The information reported here, therefore, provides evidence that the universal conservation of the P4 structure is based in part on optimization of metal ion interactions important for catalysis.     

A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction

Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5′-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5′-exon intermediate in self splicing to remain bound subsequent to 5′-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs.     

Metal ion-N7 coordination in a ribozyme branch domain by NMR

The N7 of purine nucleotides presents one of the most dominant metal ion binding sites in nucleic acids. However, the interactions between kinetically labile metal ions like Mg²⁺ and these nitrogen atoms are inherently difficult to observe in large RNAs. Rather than using the insensitive direct ¹⁵N detection, here we have used ²J-[¹H,¹⁵N]-HSQC (Heteronuclear Single Quantum Coherence) NMR experiments as a fast and efficient method to specifically observe and characterize such interactions within larger RNA constructs. Using the 27 nucleotides long branch domain of the yeast-mitochondrial group II intron ribozyme Sc.ai5γ as an example, we show that direct N7 coordination of a Mg²⁺ ion takes place in a tetraloop nucleotide. A second Mg²⁺ ion, located in the major groove at the catalytic branch site, coordinates mainly in an outer-sphere fashion to the highly conserved flanking GU wobble pairs but not to N7 of the sandwiched branch adenosine.     

Divalent hammerhead ribozyme targeting template region of human telomerase RNA has potent cleavage activity,but less inhibitory activity on telomerase

The template region of human telomerase RNA is a crucial area for regulating telomerase activity and would be a good target for ribozymes. In fact, potent telomerase inhibitory activity of the ribozyme targeting the GUC sequence of the 5(') end of this region (36-ribosome) has been well demonstrated. To search for a more potent ribozyme, we designed a divalent ribozyme to cleave both the phosphodiester bonds following the GUC and the 23 nucleotides downstream of GUA. An in vitro cleavage study showed that this divalent ribozyme cleaved telomerase RNA more efficiently than the 36-ribozyme or the 59-ribozyme to target the GUA. When this ribozyme was introduced into the carcinoma cells, its inhibitory effect on telomerase activity was less than that of the 36-ribozyme. The 59-ribozyme showed minimum activity on telomerase. This implies that, although the divalent ribozyme possesses a potent cleavage activity on hTR in vitro, the 36-ribozyme is most potent to suppress telomerase activity.     

Role of an active site guanine in hairpin ribozyme catalysis probed by exogenous nucleobase rescue

The hairpin ribozyme is a small catalytic RNA with reversible phosphodiester cleavage activity. Biochemical and structural studies exclude a requirement for divalent metal cation cofactors and implicate one active site nucleobase in particular, G8, in the catalytic mechanism. Our previous work demonstrated that the cleavage activity that is lost when G8 is replaced by an abasic residue is restored when certain nucleobases are provided in solution. The specificity and pH dependence of exogenous nucleobase rescue were consistent with several models of the rescue mechanism, including general acid base catalysis, electrostatic stabilization of negative charge in the transition state or a requirement for protonation to facilitate exogenous nucleobase binding. Detailed analyses of exogenous nucleobase rescue for both cleavage and ligation reactions now allow us to refine models of the rescue mechanism. Activity increased with increasing pH for both unmodified ribozyme reactions and unrescued reactions of abasic variants lacking G8. This similarity in pH dependence argues against a role for G8 as a general base catalyst, because G8 deprotonation could not be responsible for the pH-dependent transition in the abasic variant. Exogenous nucleobase rescue of both cleavage and ligation activity increased with decreasing pH, arguing against a role for rescuing nucleobases in general acid catalysis, because a nucleobase that contributes general acid catalysis in the cleavage pathway should provide general base catalysis in ligation. Analysis of the concentration dependence of cytosine rescue at high and low pH demonstrated that protonation promotes catalysis within the nucleobase-bound ribozyme complex but does not stabilize nucleobase binding in the ground state. These results support an electrostatic stabilization mechanism in which exogenous nucleobase binding counters negative charge that develops in the transition state.     

Assembly and activation of a kinase ribozyme

RNA activities can be regulated by modulating the relative energies of all conformations in a folding landscape; however, it is often unknown precisely how peripheral elements perturb the overall landscape in the absence of discrete alternative folds (inactive ensemble). This work explores the effects of sequence and secondary structure in governing kinase ribozyme activity. Kin.46 catalyzes thiophosphoryl transfer from ATPγS onto the 5′ hydroxyl of polynucleotide substrates, and is regulated 10,000-fold by annealing an effector oligonucleotide to form activator helix P4. Transfer kinetics for an extensive series of ribozyme variants identified several dispensable internal single-stranded segments, in addition to a potential pseudoknot at the active site between segments J1/4 and J3/2 that is partially supported by compensatory rescue. Standard allosteric mechanisms were ruled out, such as formation of discrete repressive structures or docking P4 into the rest of the ribozyme via backbone 2′ hydroxyls. Instead, P4 serves both to complete an important structural element (100-fold contribution to the reaction relative to a P4-deleted variant) and to mitigate nonspecific, inhibitory effects of the single-stranded tail (an additional 100-fold contribution to the apparent rate constant, k_obs). Thermodynamic activation parameters ΔH^‡ and ΔS^‡, calculated from the temperature dependence of k_obs, varied with tail length and sequence. Inhibitory effects of the unpaired tail are largely enthalpic for short tails and are both enthalpic and entropic for longer tails. These results refine the structural view of this kinase ribozyme and highlight the importance of nonspecific ensemble effects in conformational regulation by peripheral elements.     

Inhibiting apoptosis of CTLL-2 cells to enhance their GVL effects via anti-Fas ribozyme

Donor lymphocyte infusion (DLI) is an adoptiveimmunotherapy to achieve particular therapy aims forpatients accepting allogenetic hemopoietic stem celltransplantation [1–3]. Recently, many researches havetestified that the graft-versus-leukemia effect (GV…     

Identification of the catalytic subdomain of the VS ribozyme and evidence for remarkable sequence tolerance in the active site loop

We show here that the ribozyme domain of the Neurospora VS ribozyme consists of separable upper and lower subdomains. Deletion analysis demonstrates that the entire upper subdomain (helices III/IV/V) is dispensable for site-specific cleavage activity, providing experimental evidence that the active site is contained within the lower subdomain and within the substrate itself. We demonstrate an important role in cleavage activity for a region of helix VI called the 730 loop. Surprisingly, several loop sequences, sizes, and structures at this position can support site-specific cleavage, suggesting that a variety of non-Watson-Crick structures, rather than a specific loop structure, in this region of the ribozyme can contribute to formation of the active site.