Linkage between proton binding and folding in RNA: implications for RNA catalysis.

Small ribozymes use their nucleobases to catalyse phosphodiester bond cleavage. The hepatitis delta virus ribozyme employs C75 as a general acid to protonate the 5'-bridging oxygen leaving group, and to accomplish this task efficiently, it shifts its pKa towards neutrality. Simulations and thermodynamic experiments implicate linkage between folding and protonation in nucleobase pKa shifting. Even small oligonucleotides are shown to fold in a highly co-operative manner, although they do so in a context-specific fashion. Linkage between protonation and co-operativity of folding may drive pKa shifting and provide for enhanced function in RNA.

Mechanistic characterization of the HDV genomic ribozyme: assessing the catalytic and structural contributions of divalent metal ions within a multichannel reaction mechanism.

Hepatitis delta virus (HDV) uses genomic and antigenomic ribozymes in its replication cycle. We examined ribozyme self-cleavage over eight orders of magnitude of Mg(2+) concentration, from approximately 10(-9) to 10(-1) M. These experiments were carried out in 1 M NaCl to aid folding of the ribozyme and to control the ionic strength. The concentration of free Mg(2+) ions was established using an EDTA-Mg(2+) buffered system. Over the pH range of 5-9, the rate was independent of Mg(2+) concentration up to 10(-7) M, and of the addition of a large excess of EDTA. This suggests that in the presence of 1 M NaCl, the ribozyme can fold and cleave without using divalent metal ions. Brønsted analysis under these reaction conditions suggests that solvent and hydroxide ions may play important roles as general base and specific base catalysts. The observed rate constant displayed a log-linear dependence on intermediate Mg(2+) concentration from approximately 10(-7) to 10(-4) M. These data combined with the shape of the pH profile under these conditions are consistent with the binding of at least one structural divalent metal ion that does not participate in catalysis and binds tighter at lower pH. No evidence for a catalytic role for Mg(2+) was found at low or intermediate Mg(2+) concentrations. Addition of Mg(2+) to physiological and higher concentrations, from 10(-3) to 10(-1) M, revealed a second saturable divalent metal ion which binds tighter at high pH. The shape of the pH profile is inverted relative to that at low Mg(2+) concentrations, consistent with a general acid-base catalysis mechanism in which a cytosine (C75) acts as the general acid and a hydroxide ion from the divalent metal ion, or possibly from solvent, acts as the base. Overall, the data support a model in which the HDV ribozyme can self-cleave by multiple divalent ion-independent and -dependent channels, and in which the contribution of Mg(2+) to catalysis is modest at approximately 25-fold. Surface electrostatic potential maps were calculated on the self-cleaved form of the ribozyme using the nonlinear Poisson-Boltzmann equation. These calculations revealed several patches of high negative potential, one of which is present in a cleft near N4 of C75. These calculations suggest that distinct catalytic and structural metal ion sites exist on the ribozyme, and that the negative potential at the active site may help shift the pK(a) for N3 of C75 toward neutrality.

Efficient construction of long DNA duplexes with internal non-Watson-Crick motifs and modifications.

We have developed a semi-synthetic approach for preparing long stretches of DNA (>100 bp) containing internal chemical modifications and/or non-Watson-Crick structural motifs which relies on splint-free, cell-free DNA ligations and recycling of side-products by non-PCR thermal cycling. A double-stranded DNA PCR fragment containing a polylinker in its middle is digested with two restriction enzymes and a small insert ( approximately 20 bp) containing the modification or non-Watson-Crick motif of interest is introduced into the middle. Incorrect products are recycled to starting materials by digestion with appropriate restriction enzymes, while the correct product is resistant to digestion since it does not contain these restriction sites. This semi-synthetic approach offers several advantages over DNA splint-mediated ligations, including fewer steps, substantially higher yields ( approximately 60% overall yield) and ease of use. This method has numerous potential applications, including the introduction of modifications such as fluorophores and cross-linking agents into DNA, controlling the shape of DNA on a large scale and the study of non-sequence-specific nucleic acid-protein interactions.

Straightening of bulged RNA by the double-stranded RNA-binding domain from the protein kinase PKR.

The human interferon-induced protein kinase, PKR, is an antiviral agent that is activated by long stretches of double-stranded (ds)RNA. PKR has an N-terminal dsRNA-binding domain that contains two tandem copies of the dsRNA-binding motif and interacts with dsRNA in a nonsequence-specific fashion. Surprisingly, PKR can be regulated by certain viral and cellular RNAs containing non-Watson-Crick features. We found that RNAs containing bulges in the middle of a helix can bind to p20, a C-terminal truncated PKR containing the dsRNA-binding domain. Bulges are known to change the global geometry of RNA by bending the helical axis; therefore, we investigated the conformational changes of bulged RNA caused by PKR binding. A 66-mer DNA-RNA(+/- A(3) bulge)-DNA chimera was constructed and annealed to a complementary RNA strand. This duplex forces the protein to bind in the middle. A 66-mer duplex with a top strand composed of DNA-DNA(+/-A(3) bulge)-RNA was used as a control. Gel mobility-shift changes among the RNA-protein complexes are consistent with straightening of bulged RNA on protein binding. In addition, a van't Hoff analysis of p20 binding to bulged RNA reveals a favorable DeltaDeltaH degrees and an unfavorable DeltaDeltaS degrees relative to binding to straight dsRNA. These thermodynamic parameters are in good agreement with predictions from a nearest-neighbor analysis for RNA straightening and support a model in which the helical junction flanking the bulge stacks on protein binding. The ability of dsRNA-binding motif proteins to recognize and straighten bent RNA has implications for modulating the topology of RNAs in vivo.

A role for upstream RNA structure in facilitating the catalytic fold of the genomic hepatitis delta virus ribozyme.

Hepatitis delta virus (HDV) has a circular RNA genome that replicates by a double rolling-circle mechanism. The genomic and antigenomic versions of HDV contain a ribozyme that undergoes cis-cleavage, thereby processing the transcript into unit-length monomers. A genomic HDV transcript containing 30 nucleotides immediately upstream of the cleavage site was found to have attenuated self-cleavage. Structure mapping and site-directed mutagenesis revealed an inhibitory stretch consisting of upstream nucleotides -24 to -15 that forms a long-range pairing, termed Alt 1, with the 3' strand of P2 (P2(3')) located at the very 3'-end of the ribozyme. Two other alternative pairings were found, Alt 2, which involves upstream nucleotide-ribozyme interactions, and Alt 3, which involves ribozyme-ribozyme interactions. Self-cleavage was rescued 2700 to 20,000-fold by adding DNA oligomers, which sequester the -24/-15 inhibitory stretch in trans. Surprisingly, co-transcriptional self-cleavage occurs when the number of upstream nucleotides is increased to 54. Computer prediction and structure mapping support the existence of an unusually stable upstream hairpin involving nucleotides -54 to -18, termed P(-1)/L(-1), which sequesters the majority of the -24/-15 inhibitory stretch in cis. This hairpin is followed by a stretch of single-stranded pyrimidine-rich nucleotides, termed J(-1/1). Sequence comparison suggests that the P(-1)/L(-1)/J(-1/1) motif is conserved among known genomic HDV isolates, and that the J(-1/1) stretch is conserved among antigenomic HDV isolates. Lastly, the secondary structure of the Alt 1-containing ribozyme provides insight into possible folding intermediates of the ribozyme.

General acid-base catalysis in the mechanism of a hepatitis delta virus ribozyme.

Many protein enzymes use general acid-base catalysis as a way to increase reaction rates. The amino acid histidine is optimized for this function because it has a pK(a) (where K(a) is the acid dissociation constant) near physiological pH. The RNA enzyme (ribozyme) from hepatitis delta virus catalyzes self-cleavage of a phosphodiester bond. Reactivity-pH profiles in monovalent or divalent cations, as well as distance to the leaving-group oxygen, implicate cytosine 75 (C75) of the ribozyme as the general acid and ribozyme-bound hydrated metal hydroxide as the general base in the self-cleavage reaction. Moreover, C75 has a pK(a) perturbed to neutrality, making it "histidine-like." Anticooperative interaction is observed between protonated C75 and a metal ion, which serves to modulate the pK(a) of C75. General acid-base catalysis expands the catalytic repertoire of RNA and may provide improved rate acceleration.

Isolation and characterization of thermodynamically stable and unstable RNA hairpins from a triloop combinatorial library.

Hairpins are the most common elements of RNA secondary structure, playing important roles in RNA tertiary architecture and forming protein binding sites. Triloops are common in a variety of naturally occurring RNA hairpins, but little is known about their thermodynamic stability. Reported here are the sequences and thermodynamic parameters for a variety of stable and unstable triloop hairpins. Temperature gradient gel electrophoresis (TGGE) can be used to separate a simple RNA combinatorial library based on thermal stability [Bevilacqua, J. M., and Bevilacqua, P. C. (1998) Biochemistry 45, 15877-15884]. Here we introduce the application of TGGE to separating and analyzing a complex RNA combinatorial library based on thermal stability, using an RNA triloop library. Several rounds of in vitro selection of an RNA triloop library were carried out using TGGE, and preferences for exceptionally stable and unstable closing base pairs and loop sequences were identified. For stable hairpins, the most common closing base pair is CG, and U-rich loop sequences are preferred. Closing base pairs of GC and UA result in moderately stable hairpins when combined with a stable loop sequence. For unstable hairpins, the most common closing base pairs are AU and UG, and U-rich loop sequences are no longer preferred. In general, the contributions of the closing base pair and loop sequence to overall hairpin stability appear to be additive. Thermodynamic parameters for individual hairpins determined by UV melting are generally consistent with outcomes from selection experiments, with hairpins containing a CG closing base pair having a DeltaDeltaG degrees (37) 2.1-2.5 kcal/mol more favorable than hairpins with other closing base pairs. Sequences and thermodynamic rules for triloop hairpins should aid in RNA structure prediction and determination of whether naturally occurring triloop hairpins are thermodynamically stable.

Thermodynamic analysis of an RNA combinatorial library contained in a short hairpin.

Prediction of nucleic acid structure from sequence requires thermodynamic parameters for a variety of motifs, many of which are complex and consist of a large number of possible sequence combinations. Here we report an experimental approach for identifying the stable and unstable members of an RNA combinatorial library. Short model RNA hairpins consisting of 13 base pairs (bp) flanked by primer binding sites are constructed and separated according to their relative thermodynamic stabilities using temperature gradient gel electrophoresis (TGGE). Partially denaturing TGGE is carried out with potassium chloride, sodium chloride, or magnesium chloride salts in the gel. The TMs of model hairpins can be tuned by adjusting the concentration of urea in the gel while maintaining the correct order of stabilities for the hairpins. Mixtures of RNAs differing by a single Watson-Crick base pair are resolved according to their relative thermodynamic stabilities, as are mixtures of GC or AU base pair transversions differing in DeltaG degrees37 by only 0.3-0.5 kcal/mol. In addition, a simple combinatorial library with one position of randomization opposite a guanosine is prepared and separated into its four members by parallel and perpendicular TGGE. The order of thermodynamic stabilities for the library determined by TGGE is shown to be the same when assayed by UV-melting experiments. Analysis of the thermodynamics of folding of combinatorial libraries is general and may be applied to a wide variety of complex nucleic acid secondary and tertiary motifs in order to identify the stable and unstable members.

Thermodynamic stability of the P4-P6 domain RNA tertiary structure measured by temperature gradient gel electrophoresis.

The P4-P6 domain RNA from the Tetrahymena self-splicing group I intron is an independent unit of tertiary structure that, in the kinetic folding pathway, folds before the rest of the intron and then stabilizes the remainder of the intron's tertiary structure. We have employed temperature gradient gel electrophoresis (TGGE) to examine the unfolding of the tertiary structure of P4-P6. In 0.9 mM Mg2+, the global tertiary fold of the molecule has a melting temperature of approximately 40 degreesC and is completely unfolded by 60 degreesC. Calculated thermodynamic parameters for folding of P4-P6 are DeltaH degrees' = -28 +/- 3 kcal/mol and DeltaS degrees' = -91 +/- 8 eu under these conditions. Chemical probing of the P4-P6 tertiary structure using dimethyl sulfate and CMCT confirms that these TGGE experiments monitor the unfolding of the global tertiary fold of the domain and that the secondary structure is largely unaffected over this temperature range. Thus, unlike the entropically driven P1 docking and guanosine binding steps of Tetrahymenagroup I intron self-splicing, which have positive or zero DeltaH terms, P4-P6 tertiary structure formation is stabilized by a negative DeltaH term. This implies that enthalpically favorable hydrogen bond formation, nucleotide base stacking, and/or binding of Mg2+ within the folded structure are responsible for stabilizing the P4-P6 domain.

Binding of the protein kinase PKR to RNAs with secondary structure defects: role of the tandem A-G mismatch and noncontiguous helixes.

The human interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an antiviral agent that is activated by long stretches of dsRNA. PKR can also be activated or repressed by a series of cellular and viral RNAs containing non-Watson-Crick motifs. PKR has a dsRNA-binding domain (dsRBD) that contains two tandem copies of the dsRNA-binding motif (dsRBM). In vitro selection experiments were carried out to search for RNAs capable of binding to a truncated version of PKR containing the dsRBD. RNA ligands were selected by binding to His6-tagged proteins and chromatography on nickel(II) nitrilotriacetic acid agarose. A series of RNAs was selected that bind either similar to or tighter than a model dsRNA stem loop. Examination of these RNAs by a variety of methods, including sequence comparison, free-energy minimization, structure mapping, boundary experiments, site-directed mutagenesis, and footprinting, revealed protein-binding sites composed of noncontiguous helices. In addition, selected RNAs contained tandem A-G mismatches (5'AG3'/3'GA5'), yet bound to the truncated protein with affinities similar to duplexes containing only Watson-Crick base pairs. The NMR structure of the tandem A-G mismatch in an RNA helix (rGGCAGGCC)2 reveals a global A-form helix with minor perturbations at the mismatch [Wu, M., SantaLucia, J., Jr., and Turner, D. H. (1997) Biochemistry 36, 4449-4460]. This supports the notion that dsRBM-containing proteins can bind to RNAs with secondary structure defects as long as the RNA has an overall A-form geometry. In addition, selected RNAs are able to activate or repress wild-type PKR autophosphorylation as well as its phosphorylation of protein synthesis initiation factor eIF-2, suggesting full-length PKR can bind to and be regulated by RNAs containing a tandem A-G mismatch.

Minor-groove recognition of double-stranded RNA by the double-stranded RNA-binding domain from the RNA-activated protein kinase PKR.

The human double-stranded RNA- (dsRNA) activated protein kinase (PKR) has a dsRNA-binding domain (dsRBD) that contains two tandem copies of the dsRNA-binding motif (dsRBM). The minimal-length polypeptide required to bind dsRNA contains both dsRBMs, as determined by mobility-shift and filter-binding assays. Mobility-shift experiments indicate binding requires a minimum of 16 base pairs of dsRNA, while a minimal-length site for saturation of longer RNAs is 11 base pairs. Bulge defects in the helix disfavor binding, and single-stranded tails do not strongly influence the dsRNA length requirement. These polypeptides do not bind an RNA-DNA hybrid duplex or dsDNA as judged by either mobility-shift or competition experiments, suggesting 2'-OH contacts on both strands of the duplex stabilize binding. Related experiments on chimeric duplexes in which specific sets of 2'-OHs are substituted with 2'-H or 2'-OCH3 reveal that the 2'-OHs required for binding are located along the entire 11 basepair site. These results are supported by Fe(II) EDTA footprinting experiments that show protein-dependent protection of the minor groove of dsRNA. The dependence of dsRNA-protein binding on salt concentration suggests that only one ionic contact is made between the protein and dsRNA phosphate backbone and that at physiological salt concentrations 90% of the free energy of binding is nonelectrostatic. Thus, the specificity of PKR for dsRNA over RNA-DNA hybrids and dsDNA is largely due to molecular recognition of a network of 2'-OHs involving both strands of dsRNA and present along the entire 11 base-pair site.

A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme.

A simple model system is described which mimics the second step of splicing and reverse cyclization reactions of the self-splicing intron from Tetrahymena thermophila. This model is based on the L-21 Sca I catalyzed ligation reaction between exogenously added oligomers: cucu + UCGa L-21 Sca I cucua + UCG. Steady-state kinetics for the forward and reverse direction were measured at 15 degrees C to find oligonucleotides that exhibit Michaelis-Menten behavior with acceptable KMS. CUCU and UCGA fit both criteria and were chosen for further studies. Steady-state kinetics reveal a lag that appears to be an RNA folding step that is eliminated by preincubation of the ribozyme with 2 mM and higher [Mg2+] and by UCGA. At constant ionic strength, the Mg2+ dependence of steady-state rates exhibits a sharp maximum near 5 mM Mg2+. Pre-steady-state and steady-state kinetics, along with active-site titrations, explain the Mg2+ profile: the rate of reaction up to and including chemistry increases with Mg2+ concentration, while the fraction of active ribozyme and the rate of postchemistry steps decrease with Mg2+ concentration. The rate-limiting step at 5 mM Mg2+ for the reaction mimicking the second step of splicing is either chemistry or a conformational change before chemistry involving ribozyme bound with substrates. The rate-limiting step at 50 mM Mg2+ appears to be a postchemistry conformational change of the ribozyme or product release. At 50 mM Mg2+, single-turnover experiments support ordered binding of substrates with 5'-exon mimic binding before 3'-splice site mimic. Moreover, the 3'-splice site mimic binds and reacts in the presence of 5'-exon mimics predocked into the catalytic core. Results also indicate that Mg2+ ions associate with the ribozyme upon docking.

Thermodynamic and activation parameters for binding of a pyrene-labeled substrate by the Tetrahymena ribozyme: docking is not diffusion-controlled and is driven by a favorable entropy change.

Association and dissociation rates for the pyrene-(pyr)-labeled oligoribonucleotide substrate pyrCUCU binding to the L-21 ScaI group I ribozyme are reported as a function of temperature. Combined with thermodynamic parameters for binding of pyrCUCU to rGGAGAA, the results allow calculation of the activation and thermodynamic parameters for docking of pyrCUCU into the catalytic core of the ribozyme. The activation enthalpy for docking is 22 kcal/mol, much larger than the approximately 4 kcal/mol expected for a diffusion-controlled process. Thus, docking is not diffusion-controlled. The activation and equilibrium entropies for docking are favorable at 21 and 37 eu, respectively. The results suggest the rate-limiting step and the driving force for docking may involve desolvation of RNA functional groups or of Mg2+ ions.

Fluorescence-detected stopped flow with a pyrene labeled substrate reveals that guanosine facilitates docking of the 5' cleavage site into a high free energy binding mode in the Tetrahymena ribozyme.

Fluorescence-detected stopped flow kinetics are reported for binding of pyrene (pyr) labeled oligonucleotide substrates, pyrCUCUA and pyrCCUCUA, to the L-21 ScaI ribozyme from Tetrahymena thermophila. Both oligomer substrates contain a UA sequence that mimics the cleavage site where pG attacks the self-splicing group I intron from which the ribozyme was derived. Kinetics were measured in the presence and absence of saturating 5'-monophosphate guanosine substrate (pG) at 5 mM Mg2+ and 15 degrees C. In the absence of pG, binding of both oligonucleotide substrates is consistent with a one step mechanism involving only base pairing. Upon addition of pG, pyrCCUCUA is observed to bind in two steps: base pairing to the ribozyme to form the P1 helix and, presumably, subsequent docking of the P1 helix into the catalytic core. A third transient is also observed, which likely includes the chemical step following docking. All rate constants are measured for this mechanism. Surprisingly, the equilibrium constant for docking, K2, is unfavorable in the absence of pG (K2 < 1) and only modestly favorable in the presence of pG (K2 = 4). These results contrast with those for a 5' exon mimic, pyrCCUCU, in which docking is strongly favored under the above conditions in the absence of pG; K2 = 100 [Bevilacqua, P. C., Kierzek, R., Johnson, K. A., & Turner, D. H. (1992) Science 258, 1355-1358]. These results suggest an unfavorable interaction between the ribozyme and the pA at the site of cleavage. Implications are discussed for the catalytic strategy of the ribozyme and for the self-splicing cascade that occurs in nature.

Cooperative and anticooperative binding to a ribozyme.

The effects of guanosine 5'-monophosphate and 2'-deoxyguanosine 5'-monophosphate on the thermodynamics and kinetics of pyrene-labeled 5' exon mimic (pyCUCU) binding to the catalytic RNA (ribozyme) from Tetrahymena thermophila have been determined by fluorescence titration and kinetics experiments at 15 degrees C. pyCUCU binding to L-21 Sca I-truncated ribozyme is weaker by a factor of 5 in the presence of saturating guanosine 5'-monophosphate, whereas it is 4-fold stronger in the presence of saturating 2'-deoxyguanosine 5'-monophosphate. Results from kinetics experiments suggest that anticooperative effects in the presence of guanosine 5'-monophosphate arise primarily from slower formation of tertiary contacts between the catalytic core of the ribozyme and the P1 duplex formed by pyCUCU and GGAGGG of the ribozyme. Conversely, cooperative effects in the presence of 2'-deoxyguanosine 5'-monophosphate arise primarily from slower disruption of tertiary contacts between the catalytic core of the ribozyme and the P1 duplex. Additional experiments suggest that these cooperative and anticooperative effects are not a function of the pyrene label, are not caused by a salt effect, and are not specific to one renaturation procedure for the ribozyme.

Dynamics of ribozyme binding of substrate revealed by fluorescence-detected stopped-flow methods.

Fluorescence-detected stopped-flow and equilibrium methods have been used to study the mechanism for binding of pyrene (pyr)-labeled RNA oligomer substrates to the ribozyme (catalytic RNA) from Tetrahymena thermophila. The fluorescence of these substrates increases up to 25-fold on binding to the ribozyme. Stopped-flow experiments provide evidence that pyr experiences at least three different microenvironments during the binding process. A minimal mechanism is presented in which substrate initially base pairs to ribozyme and subsequently forms tertiary contacts in an RNA folding step. All four microscopic rate constants are measured for ribozyme binding of pyrCCUCU.

Comparison of binding of mixed ribose-deoxyribose analogues of CUCU to a ribozyme and to GGAGAA by equilibrium dialysis: evidence for ribozyme specific interactions with 2' OH groups.

Dissociation constants at 15 degrees C were measured by equilibrium dialysis for the binding of rCrUrCrU, dCrUrCrU, rCdUrCrU, rCrUdCrU, and rCrUrCdU to the L-21 ScaI form of the self-splicing group I LSU intron from Tetrahymena thermophila. Substitution of deoxyribose for ribose in each of the middle two positions makes the free energy change for binding 1-2 kcal/mol less favorable, compared to about 0.3 kcal/mol less favorable for each of the terminal positions. Dissociation constants for binding of the same oligomers to rGGAGAA were measured by optical melting methods. Substitution of a single deoxyribose for ribose makes the free energy change for binding less favorable by 0.4-0.9 kcal/mol for this simple duplex formation. Comparison of the effects for binding to ribozyme and to rGGAGAA indicate that ribozyme-specific tertiary interactions dependent on the middle two 2' OH groups of rCrUrCrU add about 2 kcal/mol of favorable free energy for binding to L-21 ScaI. Comparisons are made with results from gel retardation studies [Pyle, A. M., McSwiggen, J. A., & Cech, T. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8187-8191; Pyle, A. M., & Cech, T. R. (1991) Nature (London) 350, 628-631].

Effects of substrate structure on the kinetics of circle opening reactions of the self-splicing intervening sequence from Tetrahymena thermophila: evidence for substrate and Mg2+ binding interactions.

The self-splicing intervening sequence from the precursor rRNA of Tetrahymena thermophila cyclizes to form a covalently closed circle. This circle can be reopened by reaction with oligonucleotides or water. The kinetics of circle opening as a function of substrate and Mg2+ concentrations have been measured for dCrU, rCdU, dCdT, and H2O addition. Comparisons with previous results for rCrU suggest: (1) the 2' OH of the 5' sugar of a dinucleoside phosphate is involved in substrate binding, and (2) the 2' OH of the 3' sugar of a dimer substrate is involved in Mg2+ binding. Evidently, the binding site for a required Mg2+ ion is dependent on both the ribozyme and the dimer substrate. The apparent activation energy and entropy for circle opening by hydrolysis are 31 kcal/mol and 50 eu, respectively. The large, positive activation entropy suggests a partial unfolding of the ribozyme is required for reaction.