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D H Turner

Publications and source records attributed to D H Turner.

At least 55 records · Page 3Linked to original sources
PubMed1996

Thermodynamics of coaxially stacked helixes with GA and CC mismatches.

The thermodynamics of RNA helix-helix interfaces with intervening single- and tandem-GA or single-CC mismatches were studied by UV melting experiments. The model system consists of a hairpin with a four- or five-nucleotide 5' overhang which is bound by a short oligomer, creating the helical interface. Single GA interfaces are found to have favorable free energy increments of about 2 kcal/mol. This is similar to those reported for coaxially stacked flush interfaces of AU base pairs [Walter A. E., & Turner, D. H. (1994) Biochemistry 33, 12715-12719]. The free energy increment of the GA mismatches depends little on the sequence of the closing base pairs of the helixes, whether the break in the phosphate backbone is 5' or 3' with respect to the mismatch or whether the chains are extended beyond the helix-helix interface. Surprisingly, interfaces with single-CC mismatches have favorable free energy increments similar to those of GA interfaces, even though CC mismatches in coaxial stacks occur much less frequently in known RNA secondary structures. The results provide experimental support for the assumption that a bonus free energy is required for coaxially stacked helixes with intervening GA mismatches when free energy minimization is used to predict RNA secondary structures.

Base Composition↗
PubMed1996

Solution structure of (rGCGGACGC)2 by two-dimensional NMR and the iterative relaxation matrix approach.

The three-dimensional solution structure of the RNA self-complementary duplex [sequence: see text] was derived from two-dimensional NMR and the iterative relaxation matrix approach. Each GA mismatch forms two hydrogen bonds: A-NH6 to G-O6 and A-N1 to G-NH1 (imino). This is the first three-dimensional RNA structure with imino hydrogen-bonded tandem GA mismatches. This GA structure is totally different from the sheared tandem GA structure in [sequence: see text] which also has two hydrogen bonds: A-N7 to G-NH2 and A-NH6 to G-N3 [SantaLucia, J., Jr., & Turner, D. H. (1993) Biochemistry 32, 12612-12623]. In particular, the sheared and imino GA mismatches produce a narrowing and widening of the backbone, respectively. The results show that substitutions of Watson-Crick base pairs can have dramatic effects on the three-dimensional structures of adjacent non-Watson-Crick paired regions; i.e., the structure depends on sequence context. Thus compensating substitutions in site-directed mutagenesis experiments may not always restore biological activities.

Base Sequence↗
PubMed1996

Structural features of a six-nucleotide RNA hairpin loop found in ribosomal RNA.

The hairpin loop GUAAUA occurs frequently in ribosomal RNA. Optical melting studies show that r(GGCGUAAUAGCC) folds into a hairpin containing this loop. The structural features of the r(GGCGUAAUAGCC) hairpin have been determined by NMR and molecular modeling. NOEs from G4-H1' to A9-H2 and from A9-H2 to G10-H1' show that G4 and A9 form a sheared base pair with two hydrogen bonds: A-N7 to G-NH2 and A-NH6 to G-N3. One-dimensional NOE data show no NOEs between the imino protons of U5 and U8, but NOEs are observed between the U5-H1' and the U8-H6 and U8-H5, thus orienting the U8 imino proton away from U5. Thus U5 and U8 do not form an imino hydrogen-bonded U.U pair. The U5-H2' exhibits NOEs to both the A6-H8 and A7-H8, and the 3' phosphorus resonances of U5 and A6 are shifted downfield. This suggests that the helix turn is between the U5 and A6 nucleotides. The JH1'-H2 and JH3'-H4' coupling constants indicate that the loop is dynamic, particularly at 35 degrees C, well below the melting temperature of 63 degrees C. Structures were generated using 75 distance and 46 dihedral angle restraints. In these structures, the U5 base is stacked on the sheared base pair formed by G4 and A9 and can initiate a uridine turn similar to that observed in the anticodon loop of tRNA. The A6, A7, and U8 bases can stack on one another with their hydrogen-bonding surfaces exposed to the solvent, suggesting that they are available for tertiary interactions or protein recognition in rRNA. A range of loop structures are consistent with the data, however. The lack of formation of a U.U mismatch is consistent with a recent model that predicts the stability of hairpin loops of six nucleotides on the basis of the closing base pair and first mismatch in the loop [Serra, M. J., Axenson, T. J., & Turner, D. H. (1994) Biochemistry 33, 14289-14296].

Base Composition↗
PubMed1996

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.

Animals↗
PubMed1996

Thermodynamics of base pairing.

The study of the thermodynamics of base pairing is a rapidly expanding field due to its importance for taking advantage of opportunities presented by the growing database of nucleic acid sequences. Both natural and unnatural nucleic acids are being studied. The determination of nearest-neighbor parameters from optical melting data for RNA-DNA and DNA-DNA duplexes with Watson-Crick base pairs is particularly noteworthy. The foundations are also being laid for a theoretical understanding of these thermodynamics.

Base Composition↗
PubMed1995

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.

Animals↗
PubMed1995

The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme.

L-21 ScaI ribozyme is a linear form of the self-splicing intron from the precursor of the Tetrahymena thermophila LSU intron. The time scales for tertiary folding of L-21 ScaI were investigated in two ways after bringing it from a partially denatured state at 60 degrees C and 1 mM Mg2+ to a renatured state of 15 degrees C and 10 mM Mg2+. First, formation of a catalytically active structure was monitored by measuring the kinetics of the reaction: p*CUCUA3 + G<==>p*CUCU + GA3. This reaction mimics the first step of splicing. After 1 min of folding time, the catalytic rate is roughly 10% of the rate attained after 8 h of folding. This indicates that much of the structure refolds quickly. Also, at least two time scales of folding are observed, separated by a lag time of about 30 min. To define the regions folding on various time scales, all the guanosines of L-21 ScaI were probed with kethoxal at 15 degrees C while folding was in progress. Based on folding time scales, the guanosines can be placed into at least four classes. These are guanosines that (1) are already protected at 60 degrees C in 1 mM Mg2+ or which fold immediately, (2) fold during the lag time, (3) continue to fold after 1 h, and (4) never fold. These results give insight into the folding pathway of a group I ribozyme at nucleotide resolution. This provides useful information on the regions whose foldings are important for catalytic function of the ribozyme. The method may also provide a general way to suggest regions of an RNA that may interact with each other to form tertiary structure.

Base Sequence↗
PubMed1995

A periodic table of symmetric tandem mismatches in RNA.

The stabilities and structures of a series of RNA octamers containing symmetric tandem mismatches were studied by UV melting and imino proton NMR. The free energy increments for tandem mismatch formation are found to depend upon both mismatch sequence and adjacent base pairs. The observed sequence dependence of tandem mismatch stability is UGGU > GUUG > GAAG > or = AGGA > UUUU > CAAC > or = CUUC approximately UCCU approximately CCCC approximately ACCA approximately AAAA, and the closing base pair dependence is 5'G3'C > 5'C3'G > 5'U3'A approximately 5'A3'U. These results differ from expectations based on models used in RNA folding algorithms and from the sequence dependence observed for folding of RNA hairpins. Imino proton NMR results indicate the sequence dependence is partially due to hydrogen bonding within mismatches.

Base Sequence↗
PubMed1994

A model for the stabilities of RNA hairpins based on a study of the sequence dependence of stability for hairpins of six nucleotides.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGCXUAAUYGCC, where XY is the set of 10 possible mismatch base pairs. A nearest neighbor analysis of the data indicates the free energy for loop formation at 37 C varies from 2.9 to 4.5 kcal/mol. Thermodynamic parameters are also reported for hairpin formation by RNA sequences of the type GGXGUAAUAYCC (where XY are CG, GC, AU, UA, GU, and UG), with the common naturally occurring GA first mismatch (45% of small and large subunit rRNA loops of six). These results allow the development of a model to predict the stability of RNA hairpin loops. The model includes the size of the loop, the identity of the closing base pair, the free energy increment (delta G zero 37MM) for interaction of the closing base pair with the first mismatch, and an additional stabilization term for GA and UU first mismatches. delta G zero 37L(n) = delta G zero 37i(n) + delta G zero 37MM + 0.4 (if closed by AU or UA) -0.7 (if first mismatch is GA or UU). Here delta G zero 37i(n) is the free energy for initiating a loop of n nucleotides. delta G zero 37i(n) for n = 4-9 is 4.9, 4.4, 5.0, 5.0, 5.1, and 5.2 kcal/mol, respectively. The delta G zero 37MM is derived from measurements of model duplexes with terminal mismatches. The model gives good agreement when tested against four naturally occurring hairpin sequences.

Base Sequence↗
PubMed1994

Replacement of the conserved G.U with a G-C pair at the cleavage site of the Tetrahymena ribozyme decreases binding, reactivity, and fidelity.

There is a phylogenetically conserved G.U pair at the 5'-splice site of group I introns. When this is mutagenized to a G-C pair, splicing of these introns is greatly reduced. We have used a ribozyme derived from the Tetrahymena group I intron to compare the binding and reactivity of oligonucleotides that form either a G.U or a G-C pair at this position. Ribozyme binding of oligonucleotides at 42 degrees C was measured by native gel electrophoresis and equilibrium dialysis. Binding of GGCCCUCC (C(-1)P), which base-pairs with the ribozyme guide sequence to form a G-C at the cleavage site, was 10-fold weaker than the binding of GGCCCUCU (U(-1)P), which maintains the conserved G.U pair at the cleavage site. This is surprising since a terminal G-C enhances the binding between oligonucleotides by 20-fold relative to a terminal G.U. Thermal denaturation studies indicate that C(-1)P and several analogs with deoxy substitutions bind the guide-sequence oligonucleotide, GGAGGGAAA, as strongly as they bind the ribozyme. In contrast, U(-1)P binds 240-fold more strongly to the ribozyme than to GGAGGGAAA, a difference that is decreased by deoxy substitutions. Thus, while U(-1)P binds the ribozyme through a combination of base-pairing and specific 2-OH and other tertiary interactions, C(-1)P may bind by base-pairing alone. The substrate GGCCCUCCAAAAA (C(-1)S) is cleaved 100-fold more slowly than GGCCCUCUAAAAA (U(-1)S) and also has a higher propensity to be cleaved at the wrong nucleotide position.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Sequence dependence of stability for coaxial stacking of RNA helixes with Watson-Crick base paired interfaces.

Thermodynamic parameters from UV melting studies are reported for the helix-helix interfaces of coaxially stacked helixes in RNA. The model system consists of a short oligomer binding to a four-nucleotide overhang at the end of a hairpin stem, creating the helix-helix interface. Interfaces containing Watson-Crick base pairs are approximately 1 kcal/mol more stable than the corresponding nearest neighbor interaction in an uninterrupted helix. Thus the sequence dependence of stability for coaxially stacked interfaces is similar to that for regular helixes. This provides experimental evidence for an assumption that has been shown to improve predictions of RNA secondary structure [Walter, A. E., Turner, D. H., Kim, J., Lyttle, M. H., Muller, P., Mathews, D. H., & Zuker, M. (1994) Proc. Natl. Acad. Sci. U.S.A. (in press)]. The results should also be useful for modeling three-dimensional structures of RNA.

Base Sequence↗
PubMed1994

Coaxial stacking of helixes enhances binding of oligoribonucleotides and improves predictions of RNA folding.

An RNA model system consisting of an oligomer binding to a 4-nt overhang at the 5' end of a hairpin stem provides thermodynamic parameters for helix-helix interfaces. In a sequence-dependent manner, oligomers bind up to 1000-fold more tightly adjacent to the hairpin stem than predicted for binding to a free tetramer at 37 degrees C. For the interface (/) in [formula: see text] additional free energy change, delta delta G 37 degrees, for binding is roughly the nearest-neighbor delta G 37 degrees for propagation of an uninterrupted helix of equivalent sequence, CGGC. When X and Z are omitted, the delta delta 37 degrees is even more favorable by approximately 1 kcal/mol (1 cal = 4.184J). On average, predictions of 11 RNA secondary structures improve from 67 to 74% accuracy by inclusion of similar stacking contributions.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

The stability and structure of tandem GA mismatches in RNA depend on closing base pairs.

UV melting and imino proton NMR studies show that the stabilities and structures of tandem GA mismatches in RNA are dependent upon the closing base pairs around these mismatches. Internal loops of sequence 5'XGAY3'3'YAGXS' and 5'XAGY3'3'YGAX5' in the middle of octanucleotides have a range of stabilities over 5 kcal/mol when XY is a Watson-Crick or GU pair. The order of stabilities for these internal loops is 5'-GGAC-3' > UGAG, CGAG > AGAU > UGAA > GGAU. The motifs GGAC, UGAG, and CGAG are stabilizing, while the other GA motifs are destabilizing. The GAGC motif is more stable than CAGG and CGAG, but less stable than GGAC. Chemical shifts for imino protons suggest that the G imino proton of each GA mismatch in 5'-GGAC-3', 5'-GAGC-3', and 5'-CAGG-3' [SantaLucia, J., Jr., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819] is involved in a hydrogen bond to the base A, whereas in other 5'-XGAY-3' sequences, it is not involved in a hydrogen bond to the base A.

Adenine↗
PubMed1993

Structure of (rGGCGAGCC)2 in solution from NMR and restrained molecular dynamics.

The duplex (rGGCGAGCC)2 contains tandem G x A mismatches--a common motif in the secondary structures of biological RNAs. The three-dimensional structure of (rGGCGAGCC)2 was derived using molecular dynamics and energy minimization with NMR-derived restraints for 78 interproton distances (per strand), 18 hydrogen bonds for the six Watson-Crick G x C pairs, and 26 dihedral angles (per strand). The G x A mismatch structures are similar to those observed in a DNA duplex [Li, Y., Zon, G., & Wilson, W. D. (1991) Proc. Natl. Acad. Sci. U.S.A. 80, 26-30] and an RNA hairpin [Heus, H. A., & Pardi, A. (1991) Science 253, 191-193], with hydrogen bonds from guanine 2-amino and N3 to adenine N7 and 6-amino, respectively. The other G 2-amino and A 6-amino protons are within hydrogen-bonding distance of a phosphate oxygen and 2'-oxygen, respectively. Strong interstrand A-A and G-G stacking is observed between the G x A mismatches. This contrasts with the poor stacking observed between the G x A mismatches and closing G x C base pairs. The stems are basically A-form with all bases in the anti conformation and all nonterminal sugars in the C3'-endo conformation. The structure rationalizes previous thermodynamic, circular dichroism, and imino proton NMR results and suggests tandem G x A mismatches in RNA may provide a contact site for tertiary interactions.

Base Sequence↗
PubMed1993

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.

Animals↗
PubMed1993

RNA hairpin loop stability depends on closing base pair.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXAUAAUAYCC, where X and Y are CG, GC, AU, UA, GU, or UG. A nearest neighbor analysis of the data indicates the free energy change for loop formation at 37 degrees C, delta degrees Gl,37, averages 3.4 kcal/mol for hairpin loops closed with C.G, G.C, and G.U pairs. In contrast, delta G degree l,37 averages 4.6 kcal/mol for loops closed with A.U, U.A, or U.G pairs. Thus the stability of an RNA hairpin depends on the closing base pair. The hairpin with a GA mismatch that is formed by GGCGUAAUAGCC is more stable than the corresponding hairpin with an AA mismatch. Thus hairpin stability also depends on loop sequence. These effects are not included in current algorithms for prediction of RNA structure from sequence.

Base Composition↗