Biomedical subjects

J SantaLucia

Publications and source records attributed to J SantaLucia.

At least 19 recordsLinked to original sources

PubMed2001

Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA. II. NMR solution structure.

The solution structure of the conserved 690 hairpin from Escherichia coli 16 S rRNA was determined by NMR spectroscopy. The 690 loop is located at the surface of the 30 S subunit in the platform region and has been implicated in interactions with P-site bound tRNA, E-site mRNA, S11 binding, IF3 binding, and in RNA-RNA interactions with the 790 loop of 16 S rRNA and domain IV of 23 S rRNA. The structure reveals a novel sheared type G690.U697 base-pair with a single hydrogen bond from the G690 amino to U697-04. G691 and A696 also form a sheared pair and U692 forms a U-turn with an H-bond to the A695 non-bridging phosphate oxygen. The sheared pairs and U-turn result in the continuous single-stranded stacking of five residues from 6693 to U697 with their Watson-Crick functional groups exposed in the minor groove. The overall fold of the 690 hairpin is similar to the anticodon loop of tRNA. The structure provides an explanation for chemical protection patterns in the loop upon interaction with tRNA, the 50 S subunit, and S11. In vivo genetic studies demonstrate the functional importance of the motifs observed in the solution structure of the 690 hairpin.

Base Pair Mismatch↗

PubMed2001

Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA. III. Functional analysis of the 690 loop.

An instant-evolution experiment was performed on the eight nucleotides comprising the loop region of the 690 hairpin in Escherichia coli 16 S ribosomal RNA. Positions 690 to 697 were randomly mutated and 101 unique functional mutants were isolated, sequenced and analyzed for function in vivo. Non-random nucleotide distributions were observed at each of the mutated positions except 693 and 694. Nucleotide identity significantly affected ribosome function at positions 690, 695, 696 and 697. Pyrimidines were absent at position 696 in the instant-evolution pool as were C at position 691 and G at position 697. A highly significant covariation was observed between nucleotides 690 and 697. No functional double mutants at positions 691 and 696 were obtained from the instant-evolution pool. In our NMR structure of the 690 loop, both the G690.U697 and G691.A696 form sheared hydrogen-bonded mismatches. To further examine the functional constraints between these paired nucleotides, one set of site-directed mutations was constructed at positions 690:697 and another set was constructed at positions 691:696. Functional analysis of the site-directed mutants is consistent with our instant-evolution findings and revealed constraints on the placement of specific functional groups observed in the NMR structure. Ten instant-evolution mutants were isolated that are more functional than the wild-type. Hyperactivity in these mutants correlates with a single mutation at position 693.

Base Pair Mismatch↗

PubMed2000

Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA: analysis of the stem nucleotides.

Nucleotides 680 to 710 of Escherichia coli 16 S rRNA form a distinct structural domain required for ribosome function. The goal of this study was to determine the functional significance of pairing interactions in the 690 region. Two different secondary structures were proposed for this hairpin, based on phylogenetic and chemical modification studies. To study the effect of pairing interactions in the 690 hairpin on ribosome function and to determine which of the proposed secondary structures is biologically significant, we performed an instant-evolution experiment in which the nine nucleotides that form the proposed base-pairs and dangling ends of the 690 stem were randomly mutated, and functional mutant combinations were selected. A total of 96 unique functional mutants were isolated, assayed in vivo, and sequenced. Analysis of these data revealed extensive base-pairing and stacking interactions among the mutated nucleotides. Formation of either a Watson-Crick base-pair or G.U pair between positions 688 and 699 is absolutely required for ribosome function. We also performed NMR studies of a 31-nucleotide RNA which indicate the formation of a functionally important base-pair between nucleotides 688 and 699. Formation of a second base-pair between positions 689 and 698, however, is not essential for ribosome function, but the level of ribosome function correlates with the predicted thermodynamic stability of the nucleotide pairs in these positions. The universally conserved positions G690 and U697 are generally portrayed as forming a G.U mismatch. Our data show co-variation between these positions, but do not support the hypothesis that the G690:U697 pair forms a wobble structure. NMR studies of model 14-nt and 31-nt RNAs support these findings and show that G690 and U697 are involved in unusual stacking interactions but do not form a wobble pair. Preliminary NMR structural analysis reveals that the loop portion of the 690 hairpin folds into a highly structured and novel conformation.

Base Pair Mismatch↗

PubMed2000

Unique structural and stabilizing roles for the individual pseudouridine residues in the 1920 region of Escherichia coli 23S rRNA.

The synthesis of a 5'-O-BzH-2'- O -ACE-protected pseudouridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The availability of the phosphoramidite allows for reliable and efficient syntheses of hairpin RNAs containing single or multiple pseudouridine modifications in the stem or loop regions. Five 19-nt hairpin RNAs representing the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S rRNA were synthesized with pseudouridine residues located at positions 1911, 1915 and 1917. Thermodynamic parameters, circular dichroism spectra and NMR data are presented for all five RNAs. Overall, three different structural contexts for the pseudouridine residues were examined and compared with the unmodified RNA. Our main findings are that pseudouridine modifications exhibit a range of effects on RNA stability and structure, depending on their locations. More specifically, pseudouridines in the single-stranded loop regions of the model RNAs are slightly destabilizing, whereas a pseudo-uridine at the stem-loop junction is stabilizing. Furthermore, the observed effects on stability are approximately additive when multiple pseudouridine residues are present. The possible relationship of these results to RNA function is discussed.

Base Sequence↗

PubMed2000

Thermodynamic parameters for DNA sequences with dangling ends.

The thermodynamic contributions to duplex formation of all 32 possible single-nucleotide dangling ends on a Watson-Crick pair are reported. In most instances, dangling ends are stabilizing with free energy contributions ranging from +0.48 (GT(A)) to-0.96 kcal/mol (). In comparison, Watson-Crick nearest-neighbor increments range from -0. 58 (TA/AT) to -2.24 (GC/CG) kcal/mol. Hence, in some cases, a dangling end contributes as much to duplex stability as a Watson-Crick A-T base pair. The implications of these results for DNA probe design are discussed. Analysis of the sequence dependence of dangling-end stabilities show that the nature of the closing base pair largely determines the stabilization. For a given closing base pair, however, adenine dangling ends are always more or equally as stable as the other dangling nucleotides. Moreover, 5' dangling ends are more or equally as stabilizing as their 3' counterparts. Comparison of DNA with RNA dangling-end motifs shows that DNA motifs with 5' dangling ends contribute to stability equally or more than their RNA counterparts. Conversely, RNA 3' dangling ends contribute to stability equally or more than their DNA counterparts. This data set has been incorporated into a DNA secondary structure prediction algorithm (DNA MFOLD) (http://mfold2.wustl.edu/mfold/dna/for m1.cgi) as well as a DNA hybridization prediction algorithm (HYTHERtrade mark) (http://jsl1.chem.wayne.edu/Hyther/hythermenu .html).

Base Pairing↗

PubMed1999

Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches.

Thermodynamic measurements are reported for 51 DNA duplexes with A.A, C.C, G.G, and T.T single mismatches in all possible Watson-Crick contexts. These measurements were used to test the applicability of the nearest-neighbor model and to calculate the 16 unique nearest-neighbor parameters for the 4 single like with like base mismatches next to a Watson-Crick pair. The observed trend in stabilities of mismatches at 37 degrees C is G.G > T.T approximately A.A > C.C. The observed stability trend for the closing Watson-Crick pair on the 5' side of the mismatch is G.C >/= C.G >/= A.T >/= T.A. The mismatch contribution to duplex stability ranges from -2.22 kcal/mol for GGC.GGC to +2.66 kcal/mol for ACT.ACT. The mismatch nearest-neighbor parameters predict the measured thermodynamics with average deviations of DeltaG degrees 37 = 3.3%, DeltaH degrees = 7. 4%, DeltaS degrees = 8.1%, and TM = 1.1 degrees C. The imino proton region of 1-D NMR spectra shows that G.G and T.T mismatches form hydrogen-bonded structures that vary depending on the Watson-Crick context. The data reported here combined with our previous work provide for the first time a complete set of thermodynamic parameters for molecular recognition of DNA by DNA with or without single internal mismatches. The results are useful for primer design and understanding the mechanism of triplet repeat diseases.

Base Pair Mismatch↗

PubMed1999

Statistical mechanical simulation of polymeric DNA melting with MELTSIM.

MOTIVATION: MELTSIM is a windows-based statistical mechanical program for simulating melting curves of DNAs of known sequence and genomic dimensions under different conditions of ionic strength with great accuracy. The program is useful for mapping variations of base compositions of sequences, conducting studies of denaturation, establishing appropriate conditions for hybridization and renaturation, determinations of sequence complexity, and sequence divergence. RESULTS: Good agreement is achieved between experimental and calculated melting curves of plasmid, bacterial, yeast and human DNAs. Denaturation maps that accompany the calculated curves indicate non-coding regions have a significantly lower (G+C) composition than coding regions in all species examined. Curves of partially sequenced human DNA suggest the current database may be heavily biased with coding regions, and excluding large (A+T)-rich elements. AVAILABILITY: MELTSIM 1.0 is available at: //www.uml.edu/Dept/Chem/UMLBIC/Apps/MEL TSIM/MELTSIM-1.0-Win/meltsim. zip. Melting curve plots in this paper were made with GNUPLOT 3.5, available at: http://www.cs.dartmouth.edu/gnuplot_inf o.html Contact : blake@maine.maine.edu;

Base Composition↗

PubMed1998

NMR solution structure of a DNA dodecamer containing single G.T mismatches.

The three-dimensional solution structure of the self-complementary DNA dodecamer (CGT_GACGT_TACG above GCAT_TGCAG_TGC] which contains the thermodynamically destabilizing [TG_A above AT_T] motif was determined using two-dimensional NMR spectroscopy and simulated annealing protocols. Relaxation matrix analysis methods were used to yield accurate NOE derived distance restraints. Scalar coupling constants for the sugar protons were determined by quantitative simulations of DQF-COSY cross-peaks and used to determine sugar pucker populations. Twenty refined structures starting from random geometries converged to an average pairwise root mean square deviation of 0.49 A. Back calculated NOEs give Rc and Rx factors of 0.38 and 0.088, respectively. The final structure shows that each of the single G@T mismatches form a wobble pair with two hydrogen bonds where the guanine projects into the minor groove and the thymine projects into the major groove. The incorporation of the destabilizing [TG_A above AT_T] motif has little effect on the backbone torsion angles and helical parameters compared to standard B-form duplexes, which may explain why G.T mismatches are among the most commonly observed in DNA. The structure shows that perturbations caused by a G.T mismatch extend only to its neighboring Watson-Crick base pair, thus providing a structural basis for the applicability of the nearest-neighbor model to the thermodynamics of internal G.T mismatches.

Base Pair Mismatch↗

PubMed1998

Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs.

Improved thermodynamic parameters for prediction of RNA duplex formation are derived from optical melting studies of 90 oligoribonucleotide duplexes containing only Watson-Crick base pairs. To test end or base composition effects, new sets of duplexes are included that have identical nearest neighbors, but different base compositions and therefore different ends. Duplexes with terminal GC pairs are more stable than duplexes with the same nearest neighbors but terminal AU pairs. Penalizing terminal AU base pairs by 0.45 kcal/mol relative to terminal GC base pairs significantly improves predictions of DeltaG degrees37 from a nearest-neighbor model. A physical model is suggested in which the differential treatment of AU and GC ends accounts for the dependence of the total number of Watson-Crick hydrogen bonds on the base composition of a duplex. On average, the new parameters predict DeltaG degrees37, DeltaH degrees, DeltaS degrees, and TM within 3.2%, 6.0%, 6.8%, and 1.3 degreesC, respectively. These predictions are within the limit of the model, based on experimental results for duplexes predicted to have identical thermodynamic parameters.

Base Pairing↗

PubMed1998

Nearest-neighbor thermodynamics of internal A.C mismatches in DNA: sequence dependence and pH effects.

Thermodynamics of 27 oligonucleotides with internal A.C mismatches at two different pHs were determined from UV absorbance versus temperature melting profiles. The data were combined with four literature values and used to derive nearest-neighbor parameters for all 16 trimer sequences with internal A.C mismatches at pH 7.0 and 5. 0. The results indicate that the contribution of single A.C mismatches to duplex stability is strongly dependent on the solution pH and the nearest-neighbor context. On average, the protonation of an internal A.C mismatch stabilizes the duplex by 1.39 kcal/mol for DeltaG degrees37 and 7.0 degreesC for the TM. The nearest-neighbor parameters predict DeltaG degrees37, DeltaH degrees, DeltaS degrees, and TM of oligonucleotides presented in this study with average deviations of 6.3%, 11.0%, 12.2%, and 1.8 degreesC, respectively, at pH 7.0 and 4.7%, 5.9%, 6.1%, and 1.3 degreesC, respectively, at pH 5. 0. At pH 7.0, the contribution of single A.C mismatches to helix stability ranges from 2.25 kcal/mol for TCA/AAT to 1.22 kcal/mol for GCG/CAC. At pH 5.0, however, the contribution of A+.C mismatches ranges from 1.09 kcal/mol for TCT/AAA to -0.43 kcal/mol for GCC/CAG. Implications of the results for replication fidelity and mismatch repair are discussed.

Adenine↗

PubMed1998

Effect of mismatched complementary strands and 5'-change in sequence context on the thermodynamics and structure of benzo[a]pyrene-modified oligonucleotides.

Benzo[a]pyrene (B[a]P) is a well-studied environmental carcinogen that when activated can react with DNA to form four major adducts: (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-anti-B[a]P-dG. In this study, two oligonucleotides (5'-dCCATT-GB[a]P-CTACC-3' and 5'-dCCATC-GB[a]P-CTACC-3') were prepared, each containing the four isomeric adducts, and these were hybridized to either complementary sequences or to sequences containing an A, G, or T opposite the adducted guanine. Thermal melting curves, CD, and UV spectra of each duplex were measured and compared with the unmodified counterpart. The raw and relative thermodynamic measurements were then compared which indicated that differences occur that are both adduct and sequence dependent. These differences were next compared with the in vitro DNA polymerase incorporation data and were found to be strikingly correlated. Most significantly, for all four B[a]P isomers a mismatch of an A across from the adduct resulted in the least amount of relative destabilization, while the Watson-Crick complement C showed the most; in vitro studies showed that A is the preferred base incorporated across from each isomer, while C was incorporated least often. This observed correlation suggests that one factor contributing to misincorporation at an adduct site is the thermodynamic stability of the incorporated base. Structurally, the effect of sequence context and mismatched complementary strands were also compared, suggesting that all adducts tend to intercalate within the helix when they are complemented with a mismatched complementary strand. In addition, the level of this intercalation seems to be both sequence and stereoisomer dependent.

Base Sequence↗

PubMed1998

Thermodynamics of internal C.T mismatches in DNA.

Thermodynamics of 23 oligonucleotides with internal single C.T mismatches were obtained by measuring UV absorbance as a function of temperature. Results from these 23 duplexes were combined with three measurements from the literature to derive nearest-neighbor thermodynamic parameters for seven linearly independent trimer sequences with internal C.T mismatches. The data show that the nearest-neighbor model is adequate for predicting thermodynamics of oligonucleotides with internal C.T with average deviations for Delta G degrees37, Delta H degrees, Delta S degrees and T m of 6.4%, 9.9%, 10.6%, and 1.9 degreesC respectively. C.T mismatches destabilize the duplex in all sequence contexts. The thermodynamic contribution of C. T mismatches to duplex stability varies weakly depending on the orientation of the mismatch and its context and ranges from +1.02 kcal/mol for GCG/CTC and CCG/GTC to +1.95 kcal/mol for TCC/ATG.

Cytidine↗

PubMed1998

Nearest neighbor thermodynamic parameters for internal G.A mismatches in DNA.

Thermodynamics of 22 oligonucleotides with internal single G.A mismatches dissolved in 1 M NaCl were determined from absorbance versus temperature melting curves. These data, combined with five literature sequences, were used to derive nearest-neighbor thermodynamic parameters for seven linearly independent trimer sequences with internal G.A mismatches and Watson-Crick flanking base pairs. The G.A mismatch parameters predict DeltaG degrees 37, DeltaH degrees, DeltaS degrees, and TM with average deviations of 4.4%, 7.4%, 8.0%, and 1.5 degrees C, respectively. The nearest-neighbor parameters show that G.A mismatch stability is strongly context dependent, and DeltaG degrees 37 ranges from +1.16 kcal/mol for TGA/AAT to -0.78 kcal/mol for GGC/CAG. In addition, one-dimensional 1H NMR spectra show that the G.A pairing geometry is pH and context dependent.

Base Composition↗

PubMed1998

A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics.

A unified view of polymer, dumbbell, and oligonucleotide nearest-neighbor (NN) thermodynamics is presented. DNA NN DeltaG degrees 37 parameters from seven laboratories are presented in the same format so that careful comparisons can be made. The seven studies used data from natural polymers, synthetic polymers, oligonucleotide dumbbells, and oligonucleotide duplexes to derive NN parameters; used different methods of data analysis; used different salt concentrations; and presented the NN thermodynamics in different formats. As a result of these differences, there has been much confusion regarding the NN thermodynamics of DNA polymers and oligomers. Herein I show that six of the studies are actually in remarkable agreement with one another and explanations are provided in cases where discrepancies remain. Further, a single set of parameters, derived from 108 oligonucleotide duplexes, adequately describes polymer and oligomer thermodynamics. Empirical salt dependencies are also derived for oligonucleotides and polymers.

DNA↗

PubMed1997

The crystal structure of an RNA oligomer incorporating tandem adenosine-inosine mismatches.

The X-ray crystallographic structure of the RNA duplex [r(CGCAIGCG)]2 has been refined to 2.5 A. It shows a symmetric internal loop of two non-Watson-Crick base pairs which form in the middle of the duplex. The tandem A-I/I-A pairs are related by a crystallographic two-fold axis. Both A(anti)-I(anti) mismatches are in a head-to-head conformation forming hydrogen bonds using the Watson-Crick positions. The octamer duplexes stack above one another in the cell forming a pseudo-infinite helix throughout the crystal. A hydrated calcium ion bridges between the 3'-terminal of one molecule and the backbone of another. The tandem A-I mismatches are incorporated with only minor distortion to the backbone. This is in contrast to the large helical perturbations often produced by sheared G-A pairs in RNA oligonucleotides.

Adenosine↗

PubMed1997

Thermodynamics and NMR of internal G.T mismatches in DNA.

Thermodynamics of 39 oligonucleotides with internal G.T mismatches dissolved in 1 M NaCl were determined from UV absorbance versus temperature profiles. These data were combined with literature values of six sequences to derive parameters for 10 linearly independent trimer and tetramer sequences with G.T mismatches and Watson-Crick base pairs. The G.T mismatch parameters predict DeltaG degrees 37, DeltaH degrees , DeltaS degrees , and TM with average deviations of 5.1%, 7.5%, 8.0%, and 1.4 degrees C, respectively. These predictions are within the limits of what can be expected for a nearest-neighbor model. The data show that the contribution of a single G.T mismatch to helix stability is context dependent and ranges from +1.05 kcal/mol for AGA/TTT to -1.05 kcal/mol for CGC/GTG. Several tests of the applicability of the nearest-neighbor model to G.T mismatches are described. Analysis of imino proton chemical shifts show that structural perturbations from the G.T mismatches are highly localized. One-dimensional NOE difference spectra demonstrate that G.T mismatches form stable hydrogen-bonded wobble pairs in diverse contexts. Refined nearest-neighbor parameters for Watson-Crick base pairs are also presented.

Base Composition↗

PubMed1997

In vivo determination of RNA structure-function relationships: analysis of the 790 loop in ribosomal RNA.

The 790 loop is a conserved hairpin located between positions 786 and 796 of Escherichia coli 16 S rRNA that is required for ribosome function. Using a novel genetic approach, all positions in the loop were simultaneously mutated and functional mutant sequences were selected in vivo. This "instant evolution" experiment revealed that approximately 190 of the 262,144 possible mutant sequences were functional. Analysis of functional mutant sequences allowed discrimination between nucleotides directly involved in protein synthesis and those involved primarily in loop structure. Among the functional mutant sequences, positions 789 and 791 were invariant and extensive covariation was observed among the nucleotides at the base of the loop at positions 787, 788, 794 and 795. NMR and thermodynamic analyses of model 790 hairpins in vitro revealed weak pairing interactions between positions 787 and 795 and between positions 788 and 794 consistent with the in vivo mutational analysis. Functional analysis of site-directed mutants containing all possible nucleotide combinations at positions 787 and 795 in vivo showed that stable base-pairs at these positions prevent subunit association.

Chloramphenicol↗

PubMed1997

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

The three-dimensional solution structure of the RNA self-complementary duplex [formula in 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 GA structure differs from the sheared tandem GA structure in [formula in 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], although the only difference between the two sequences is the order of the two GA mismatches. Inspection of three-dimensional structures indicates that substituting [formula in text] for [formula in text] makes GA mismatches unable to have stable sheared conformations. This may explain why the [formula in text] motif is rarely observed in nature, whereas [formula in text] common.

Crystallography, X-Ray↗