Monellin (MNEI) at 1.15 A resolution.

The X-ray crystal structure of a single-chain monellin protein (MNEI) has been determined at 1.15 A resolution. The model was refined to convergence employing anisotropic displacement parameters and riding H atoms to produce a final model with R(work) and R(free) values of 0.132 and 0.162, respectively. The crystal contains a single MNEI protein in the asymmetric unit and unusually lacks the dimer interface observed in all previous crystal structures of monellin and its single-chain derivatives. The high resolution allowed a more detailed view of MNEI than previously possible, with 38 of the 96 residues modelled with alternative side-chain conformations, including four core residues Thr12, Cys41, Leu62 and Ile75. Four stably bound negative ions were also located, providing new insight into potential electrostatic interactions of MNEI with the largely negatively charged surface of the sweet taste receptor T1R2-T1R3.

Crystal structure of a conserved ribosomal protein-RNA complex.

The structure of a highly conserved complex between a 58-nucleotide domain of large subunit ribosomal RNA and the RNA-binding domain of ribosomal protein L11 has been solved at 2.8 angstrom resolution. It reveals a precisely folded RNA structure that is stabilized by extensive tertiary contacts and contains an unusually large core of stacked bases. A bulge loop base from one hairpin of the RNA is intercalated into the distorted major groove of another helix; the protein locks this tertiary interaction into place by binding to the intercalated base from the minor groove side. This direct interaction with a key ribosomal RNA tertiary interaction suggests that part of the role of L11 is to stabilize an unusual RNA fold within the ribosome.

The crystal structure of the RNA/DNA hybrid r(GAAGAGAAGC). d(GCTTCTCTTC) shows significant differences to that found in solution.

The crystal structure of the RNA/DNA hybrid r(GAAGAGAAGC). d(GCTTCTCTTC) has been solved and refined at 2.5 A resolution. The refinement procedure converged at R = 0.181 for all reflections in the range 20.0-2.5 A. In the crystal, the RNA/DNA hybrid duplex has an A' conformation with all but one of the nucleotide sugar moieties adopting a C3'- endo (N) conformation. Both strands in the double helix adopt a global conformation close to the A-form and the width of the minor groove is typical of that found in the crystal structures of other A-form duplexes. However, differences are observed between the RNA and DNA strands that make up the hybrid at the local level. In the central portion of the duplex, the RNA strand has backbone alpha, beta and gamma torsion angles that alternate between the normal gauche -/ trans / gauche + conformation and an unusual trans / trans / trans conformation. Coupled with this so-called 'alpha/gamma flipping' of the backbone torsion angles, the distance between adjacent phosphorous atoms on the RNA strand systematically varies. Neither of these phenomena are observed on the DNA strand. The structure of the RNA/DNA hybrid presented here differs significantly from that found in solution for this and other sequences. Possible reasons for these differences and their implications for the current model of RNase H activity are discussed.

A functional ribosomal RNA tertiary structure involves a base triple interaction.

Comparative sequence analysis reveals a coordinated set of nucleotide exchanges between the base pair 1092/1099 and the unpaired position 1072 [(1092/1099)1072] in the L11 binding domain of 23S ribosomal RNA. This set of exchanges has occurred at least 4 times during evolution, suggesting that these positions form a base triple. The analysis further suggests an important role for positions (1065/1073), adjacent to 1072. The covariation at positions (1092/1099)1072 is studied here by analysis of RNA variants using UV melting and binding of ribosomal protein L11 and thiostrepton to assay for tertiary folding of this domain. The tertiary structure of the RNA is eliminated by alteration of the unpaired nucleotide (C1072 to U mutation), and binding of L11 and thiostrepton are reduced 10-fold compared to the wild type. In contrast, substitution of the base pair (CG1092/1099 to UA mutation) allows formation of the tertiary structure but dramatically alters the pH dependence of tertiary folding. The fully compensated set of mutations, (CG)C to (UA)U, restores the tertiary structure of the RNA to a state almost identical to the wild type. The nature of this base triple and its implications for the folding of the RNA and ligand interactions are discussed.

RNA structure.

New information concerning RNA structure is accumulating at an ever increasing rate-from short helices with mismatched bases of 5S rRNA and complex RNA aptamers. The importance of recurring structural motifs, ion binding, and the kinetics and energetics of folding in RNA structure and function is now being recognized and addressed.

The orientation and dynamics of the C2'-OH and hydration of RNA and DNA.RNA hybrids.

The stereochemical and dynamic properties of the C2' hydroxyl group in several DNA.RNA hybrids have been measured by NMR and compared with the homologous RNA duplex. The C2'-OH NMR signals of the RNA strands were identified, and numerous specific assignments were made. The rate constants for exchange of the hydroxyl protons with water were determined at 5 degrees C, and were found to depend on both the position within a particular sequence and the nature of the duplex. On average, the exchange rate constants were slowest for the hybrids of composition rR.dY, and fastest for the RNA duplex, with an overall range of approximately 10-50/s. In the DNA.RNA hybrids, strong NOEs and ROEs were observed between the OH and the H1' of the same sugar, unambiguously showing that the OH proton points toward the H1' most of the time, and not toward the O3' of the same sugar. Evidence for significant hydration in both grooves of the DNA.RNA hybrids and the DNA duplex was found in ROESY and NOESY experiments. On average, the minor groove of the DNA.RNA hybrids showed more kinetically significant hydration than the DNA, which can be attributed to the hydrophilic lining of hydroxyl groups in RNA.

Solution structures of DNA.RNA hybrids with purine-rich and pyrimidine-rich strands: comparison with the homologous DNA and RNA duplexes.

The structures of d(GAAGAGAAGC).d(GCTTCTCTTC), d(GAAGAGAAGC). r(GCUUCUCUUC), r(GAAGAGAAGC).d(GCTTCTCTTC), and r(GAAGAGAAGC). r(GCUUCUCUUC) have been determined in solution from NMR data. Globally, the pure DNA and RNA duplexes were in the B and A forms, respectively. The two DNA.RNA hybrids were neither A nor B, but closer globally to the A than the B form. However, the thermodynamically less stable d(GAAGAGAAGC).r(GCUUCUCUUC) duplex has a significantly different conformation from r(GAAGAGAAGC). d(GCTTCTCTTC). Structures were calculated based on the NMR data, using restrained molecular dynamics. A new approach to the treatment of conformational averaging based on a prioriprobabilities has been used. The nucleotides were treated by fitting the scalar coupling data and NOE time courses to a two-state model comprising N and S sugar puckers each with a different glycosidic torsion angle, and the mole fraction of the S state. Restraint sets for different distributions of N and S states within molecules were constructed, such that each nucleotide was weighted in the ensemble according to the mole fractions (or a prioriprobabilities). The individual nucleotide conformations were strongly restrained, whereas the internucleotide restraints were set relatively loosely. Ensembles of conformations were generated and assessed by comparison of the NOEs calculated from ensemble-averaged relaxation matrices with the experimental NOEs. The ensemble averages accounted for the experimental data much better than any individual member, or for structures calculated assuming a single unique conformation. The two hybrids populated different degrees of conformational space. There was a general trend in minor and major groove widths in the order d(GAAGAGAAGC).d(GCTTCTCTTC), d(GAAGAGAAGC).r(GCUUCUCUUC), r(GAAGAGAAGC).d(GCTTCTCTTC), r(GAAGAGAAGC).r(GCUUCUCUUC) and a similar progression in global character from B-like to A-like structures. Furthermore, r(GAAGAGAAGC).d(GCTTCTCTTC) showed a greater dispersion of conformations in the ensemble than d(GAAGAGAAGC).r(GCUUCUCUUC), reflecting the greater flexibility of the sugars. If conformational averaging of the nucleotides is ignored, incorrect virtual structures are produced that nevertheless are able to satisfy a substantial fraction of the experimental data.

Comparison of the electrophoretic and hydrodynamic properties of DNA and RNA oligonucleotide duplexes.

The electrophoretic behavior of defined DNA and RNA oligonucleotide duplexes from 10 to 20 bp in length has been investigated as a function of salt conditions, gel concentration, and temperature. The RNA oligomers migrated much more slowly than the DNA oligomers of the same sequence under all conditions. From sedimentation equilibrium and velocity measurements, the apparent partial specific volume in 0.1 M KCI, 20 mM NaPi, pH 7, was determined as 0.56 +/- 0.015 ml g(-1) for DNA and 0.508 ml g(-1) for RNA. The translational friction coefficients were determined and compared with the values calculated for cylinders. Taking into account the shape factors, the solution density, and partial specific volumes, the effective degree of hydration was estimated as 0.8-1 g g(-1) DNA. There was no significant difference in the frictional coefficients of the DNA and RNA oligomers, indicating that the effective sizes of DNA and RNA are very similar in solution. The differential electrophoretic mobility of DNA and RNA must arise from the differences in interaction with counterions, which is probably a global property of the oligonucleotides.

Conformational properties and thermodynamics of the RNA duplex r(CGCAAAUUUGCG)2: comparison with the DNA analogue d(CGCAAATTTGCG)2.

The thermodynamic stability of nine dodecamers (four DNA and five RNA) of the same base composition has been compared by UV-melting. TheDeltaG of stabilisation were in the order: r(GACUGAUCAGUC)2>r(CGCAAATTTGCG)2 approximately r(CGCAUAUAUGCG)2>d(CGCAAATTTGCG)2 approximately r(CGCAAAUUUGCG)2>d(CGCATATATGCG)2 approximately d(GACTGATCAGTC)2>r(CGCUUUAAAGCG)2 approximately d(CGCTTTAAAGCG)2. Compared with the mixed sequences, both r(AAAUUU) and r(UUUAAA) are greatly destablising in RNA, whereas in DNA, d(TTTAAA) is destabilising but d(AAATTT) is stabilising, which has been attributed to the formation of a special B'structure involving large propeller twists of the A-T base pairs. The solution structure of the RNA dodecamer r(CGCAAAUUUGCG)2has been determined using NMR and restrained molecular dynamics calculations to assess the conformational reasons for its stability in comparison with d(CGCAAATTTGCG)2. The structures refined to a mean pairwise r.m.s.d. of 0.89+/-0.29 A. The nucleotide conformations are typical of the A family of structures. However, although the helix axis displacement is approximately 4.6 A into the major groove, the rise (3.0 A) and base inclination ( approximately 6 degrees ) are different from standard A form RNA. The extensive base-stacking found in the AAATTT tract of the DNA homologue that is largely responsible for the higher thermodynamic stability of the DNA duplex is reduced in the RNA structure, which may account for its low relative stability.

The dependence of DNase I activity on the conformation of oligodeoxynucleotides.

We have developed a sensitive continuous assay for nucleases using proton release. The assay has been applied to the determination of the kinetics of DNase I acting on short, defined deoxyoligonucleotides. The dependence of Kcat/K(m) on sequence and structure of short oligonucleotide substrates has been measured: increasing lengths of AnTn sequences decrease the rate of cleavage. G.A mismatches in which the bases pair using imino protons are cleaved quite effectively by DNase I. In contrast, tandem G.A mismatches which use amino pairing and have BII phosphodiesters, are refractory to DNase I. Also, the DNA strands of DNA.RNA hybrid duplexes are not cleaved by DNase I. These results show that the global conformation of a duplex and the details of its minor groove affect the cleavage efficiency by DNase I. The assay has also been used to measure the inhibition constant of the minor-groove-binding ligand propamidine. A value of 3 microM has been determined for binding to the sequence d(CGCGAATTCGCG)2, showing that dissociation constants can be determined even when there are no convenient optical signals for titrations.

Hydration of the RNA duplex r(CGCAAAUUUGCG)2 determined by NMR.

The so-called spine of hydration in the minor groove of AnTn tracts in DNA is thought to stabilise the structure, and kinetically bound water detected in the minor groove of such DNA species by NMR has been attributed to a narrow minor groove [Liepinsh, E., Leupin, W. and Otting, G. (1994) Nucleic Acids Res. 22, 2249-2254]. We report here an NMR study of hydration of an RNA dodecamer which has a wide, shallow minor groove. Complete assignments of exchangeable protons, and a large number of non-exchangeable protons in r(CGCAAAUUUGCG)2 have been obtained. In addition, ribose C2'-OH resonances have been detected, which are probably involved in hydrogen bonds. Hydration at different sites in the dodecamer has been measured using ROESY and NOESY experiments at 11.75 and 14.1 T. Base protons in both the major and minor grooves are in contact with water, with effective correlation times for the interaction of approximately 0.5 ns, indicating weak hydration, in contrast to the hydration of adenine C2H in the homologous DNA sequence. NOEs to H1' in the minor groove are consistent with hydration water present that is not observed in the analogous DNA sequence. Hydration kinetics in nucleic acids may be determined by chemical factors such as hydrogen-bonding more than by simple conformational factors such as groove width.

Comparison of the thermodynamic stabilities and solution conformations of DNA.RNA hybrids containing purine-rich and pyrimidine-rich strands with DNA and RNA duplexes.

The conformations and thermodynamic stabilities of duplexes containing purine-rich (GAA-GAGAAGC) and pyrimidine-rich (GCTTCTCTTC or GCUUCUCUUC) DNA and RNA strands have been measured by UV melting, electrophoresis, circular dichroism, and NMR spectroscopy. The free energies of stabilization (-delta G) were in the order rR.rY > rR.dY > dR.dY > dR.rY. The two DNA.RNA hybrid duplexes showed conformational properties intermediate between those of DNA.DNA and RNA.RNA duplexes and also different from one another. Differences between 1H chemical shifts of the DNA strands in the two hybrid duplexes and those of the DNA duplex were larger than analogous shift differences for the RNA protons, and the differences were larger for the purine than the pyrimidines in the DNA strands. Detailed analysis of the nucleotide conformations using both NOE and scalar coupling data showed that the sugar conformations of the ribonucleotides are all near C3'-endo. The deoxyribonucleotides were in the "S" domain, i.e., near C2'-endo in the DNA duplex, and C1'-exo to C2'-endo in the two hybrids. However, the deoxyriboses in the two hybrids appear more flexible than in the DNA duplex, with the fraction in the "N" (C3'-endo) state increasing in the order dR.dY < dR.rY < rR.dY. Globally, the pure DNA duplex was B-form and the pure RNA duplex A form. The two DNA.RNA hybrids were neither A nor B, but closer globally to the A than the B form. The less stable dR.rY duplex has a significantly different conformation from rR.dY both at the local nucleotide level and globally.