In order to elucidate roles of the 2'-O-methylation of pyrimidine nucleotide residues of tRNAs, conformations of 2'-O-methyluridylyl(3'----5')uridine (UmpU), 2'-O-methyluridine 3'-monophosphate (Ump), and 2'-O-methyluridine (Um) in 2H2O solution were analyzed by one- and two-dimensional proton NMR spectroscopy and compared with those of related nucleotides and nucleoside. As for UpU and UmpU, the 2'-O-methylation was found to stabilize the C3'-endo form of the 3'-nucleotidyl unit (Up-/Ump-moiety). This stabilization of the C3'-endo form is primarily due to an intraresidue effect, since the conformation of the 5'-nucleotidyl unit (-pU moiety) was only slightly affected by the 2'-O-methylation of the 3'-nucleotide unit. In fact even for Up and Ump, the 2'-O-methylation significantly stabilizes the C3'-endo form by 0.8 kcal/.mol-1. By contrast, for nucleosides (U and Um), the C3'-endo form is slightly stabilized by 0.1 kcal/.mol-1. Accordingly, the stabilization of the C3'-endo form by the 2'-O-methylation is primarily due to the steric repulsion among the 2-carbonyl group, the 2'-O-methyl group and the 3'-phosphate group in the C2'-endo form. For some tRNA species, 2-thiolation of pyrimidine residues is found in positions where the 2'-O-methylation is found for other tRNA species.(ABSTRACT TRUNCATED AT 250 WORDS)

The three-dimensional structure of a short DNA fragment, d(GCGAAGC) exhibiting an extraordinarily stable hairpin structure was determined by nuclear magnetic resonance spectroscopy. Two possible models were obtained by molecular modelling using distance and torsion constraints. Only one of these two models is the correct structure, which can clearly explain all the 1H chemical shifts. d(GCGAAGC) is folded back on itself between A4 and A5, and all the sugars in the fragment adopt the C2'-endo conformation. This compact molecule is stabilized by regular extensive base-stacking interaction within each B-form helical strand of G1C2G3A4 and AAG6CA, and by two G-C and one G3-A5 base pairs. The molecule is hard to differentiate into stem and loop regions, so that we classify it as a turn (hairpin-turn) structure exerted by a single-stranded DNA. This highly stacked structure shows high thermostability and strong resistance against nucleases contained in E.coli extracts and in human serum.

The backbone 1H, 13C, and 15N resonances of the c-Ha-Ras protein [a truncated version consisting of residues 1-171, Ras(1-171)] bound with GMPPNP (a slowly hydrolyzable analogue of GTP) were assigned and compared with those of the GDP-bound Ras(1-171). The backbone amide resonances of amino acid residues 10-13, 21, 31-39, 57-64, and 71 of Ras(1-171).GMPPNP, but not those of Ras(1-171).GDP, were extremely broadened, whereas other residues of Ras(1-171).GMPPNP exhibited amide resonances nearly as sharp as those of Ras(1-171). GDP. The residues exhibiting the extreme broadening, except for residues 21 and 71, are localized in three functional loop regions [loops L1, L2 (switch I), and L4 (switch II)], which are involved in hydrolysis of GTP and interactions with other proteins. From the temperature and magnetic field strength dependencies of the backbone amide resonance intensities, the extreme broadening was ascribed to the exchange at an intermediate rate on the NMR time scale. It was shown that the Ras(1-171) protein bound with GTP or GTPgammaS (another slowly hydrolyzable analogue of GTP) exhibits the same type of broadening. Therefore, it is a characteristic feature of the GTP-bound form of Ras that the L1, L2, and L4 loop regions, but not other regions, are in a rather slow interconversion between two or more stable conformers. This phenomenon, termed a "regional polysterism", of these loop regions may be related with their multifunctionality: the GTP-dependent interactions with several downstream target groups such as the Raf and RalGDS families and also with the GTPase activating protein (GAP) family. In fact, the binding of Ras(1-171).GMPPNP with the Ras-binding domain (residues 51-131) of c-Raf-1 was shown to eliminate the regional polysterism nearly completely. It was indicated, therefore, that each target/regulator selects its appropriate conformer among those presented by the "polysteric" binding interface of Ras. As the downstream target groups exhibit no apparent sequence homology to each other, it is possible that one target group prefers a conformer different from that preferred by another group. The involvement of loop L1 in the regional polysterism might suggest that the negative regulators, GAPs, bind to the polysteric binding interface (loops L2 and L4) of Ras and cooperatively select a conformer suitable for transition of the GTPase catalytic center, involving loops L1 and L4, into the highly active state.