Idealized atomic coordinates of yeast phenylalanine transfer RNA.

The atomic coordinates are given for yeast phenylalanine transfer RNA in the orthorhombic crystal form. The structure has been refined by fitting to successively improved electron density maps at 2.7 Å resolution. The model fitting has been accomplished by using an interactive computer graphics system to minimize the errors inherent in manual model building and coordinate measurements, using an optical comparator. The atomic coordinates have then been “idealized” to make bond distances, bond angles, steric conformation and non-bonded contacts close to standard values, while constraining the model to fit the electron density maps.     

Molecular-dynamics simulation of phenylalanine transfer RNA. II. Amplitudes, anisotropies, and anharmonicities of atomic motions

The atomic motions from a molecular-dynamics simulation of yeast tRNA^Phe are analyzed and compared with those observed in protein simulations. In general, the tRNA motions are of larger amplitude, they are more anisotropic, and they arise from potentials of mean force that are more anharmonic than in the protein case. In both cases, the amplitudes are largest for atoms on the surface of the molecules. On the other hand, the most anisotropic and anharmonic atomic motions are generally found in the interior of the tRNA, while they are found on the surface of the protein. These differences are discussed in terms of the differences in structure between nucleic acids and proteins.     

Molecular dynamics of phenylalanine transfer RNA

The atomic motions of yeast phenylalanine transfer RNA have been simulated using the molecular dynamics algorithm. Two simulations were carried out for a period of 12 picoseconds, one with a normal Van der Waals potential and the other with a modified Van der Waals potential intended to mimic the effect of solvent. An analysis of large scale motions, surface exposure, root mean square displacements, helical oscillations and relaxation mechanisms reveals the maintenance of stability in the simulated structures and the general similarity of the various dynamic features of the two simulations. The regions of conformational flexibility and rigidity for tRNA(Phe) have been shown in a quantitative measure through this approach.     

Large-amplitude bending motions in phenylalanine transfer RNA

Conformational energy calculations on yeast tRNA^Phe reveal several likely modes of intramolecular bending, including both hingelike motions (rotations about a discrete point) and distributed flexibility (deformations that bend a double-helical segment along a smooth curve). By combining these modes of motion, the molecule can be bent from the L-shaped crystallographic structure to two extremes. It can be straightened into a nearly linear conformation at an energy cost of about 50 kcal/mol, and it can be doubled over to a conformation where the anticodon and the amino acid acceptor terminus are separated by about 40 Å at an energy cost of less than 100 kcal/mol. A bending range of over 100° can be covered for 50 kcal/mol, and we estimate that this value could be cut in half with a minimization algorithm that produced optimum stereochemistry. These energies are comparable to those that would be associated with changes in solvation due to changes in surface area as the molecule bends, indicating that there are no major steric barriers to tRNA flexibility and that variations in solvent conditions and interactions with other molecules may produce large changes in the overall conformation of tRNA.     

Strong magnesium binding sites in yeast phenylalanine transfer RNA.

To ascertain the sites that are available for strong binding between magnesium ions and phosphate groups in yeast phenylalanine transfer RNA, all distances below 5.5 A separating the phosphoryl oxygens (Op) of the 76 nucleotide residues have been computed from the latest atomic coordinates for the monoclinic form of the tRNA crystallized in the presence of magnesium chloride. The 5.5 A distance is chosen as the upper limit expected for Op....Op distances involved in strong magnesium-phosphate binding, on the basis of studies on a model magnesium phosphodiester hydrate, taking into account the quoted standard deviation in the tRNA atomic coordinates. It is concluded that there are four possible sites for strong magnesium binding in the tRNA molecule, in addition to the three sites previously reported. One of the hypothetical sites: m2G10-OL, U47-OR, could be involved in the first stage of melting of the tRNA molecule, and may be relevant to tertiary structure stabilization, since it links the dihydrouridine arm with the extra (V) loop.     

Molecular-dynamics simulation of phenylalanine transfer RNA. I. Methods and general results

A 24-ps molecular-dynamics simulation of motions in yeast tRNA^Phe has been completed. The overall structure of the molecule is well preserved, for the motions represent fluctuations about an average structure that is very much like the crystallographic structure. The four helical stems remain intact, the structures of the loop regions do not deteriorate, and even the base stacking in the single-stranded amino acid acceptor terminus is maintained. With two exceptions, none of the sugar puckers is significantly changed. The unconstrained floppy motions of base A76 are responsible for the repuckering of ribose 76. The other sugar that repuckers is ribose, 46, and this is the result of a very small structural change in the center of the molecule that is also responsible for the breakage of one tertiary hydrogen bond. This change in local structure does not seriously distort the base-stacking and intercalation patterns where the variable loop and the D-stem interact.     

Raman spectra of ten aqueous transfer RNAs and 5S RNA. Conformational comparison with yeast phenylalanine transfer RNA.

Eleven native transfer RNAs have been prepared so as to maintain their Mg2+ content. Their aqueous Raman spectra show a high, relatively constant amount of order in the ribophosphate backbone, as indicated by the ratio 1.73 +/- 0.05 for I814/I1100 in all samples. Variation in the effectiveness of stacking of guanine and adenine bases is seen, though most of the transfer RNAs studied have a comparable degree of stacking to that found in phenylalanine transfer RNA from yeast, whose tertiary structure has been determined by X-ray crystallography. The spectrum of Escherichia coli 5S RNA indicates that the stacking efficiency of the guanine bases is much higher in 5S RNA than in yeast in phenylalanine transfer RNA, while that of the adenine bases is lower.     

Nucleotide sequence of phenylalanine transfer RNA from Schizosaccharomyces pombe: implications for transfer RNA recognition by yeast phenylalanyl-tRNA synthetase.

The nucleotide sequence of Schizosaccharomyces pombe tRNAPhe was determined to be pG-U-C-G-C-A-A-U-G**-G*-U-G-psi-A-G-D-D-G-G-G-A-G-C-A-psi-G*-A-C-A-G-A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-G-U-U-G-m7G-U*-C-A-U-C-G-G-T-psi-C-G-A-U-C-C-C-G-G-U-U-U-G-U-G-A-C-A-C-C-AOH. This sequence differs from that of S. cerevisiae tRNAPhe in 27 nucleotides. Saccharomyces cerevisiae phenylalanyl-tRNA synthetase aminoacylates both the homologous tRNAPhe and S. pombe t-NAPhe; the reactions have similar Km and Vmax values. However, the nucleotide sequence in the D stem is different in the two tRNAs. This region was proposed by Roe, B., et al. [(1973) Biochemistry 12, 4146--4154] to be the major recognition site for yeast phenylalanyl-tRNA synthetase, but the present results cast doubt on the validity of this hypothesis.     

Restrained refinement of the monoclinic form of yeast phenylalanine transfer RNA. Temperature factors and dynamics, coordinated waters, and base-pair propeller twist angles

The structure of yeast phenylalanine transfer RNA in the monoclinic form has been further refined by using the restrained least-squares method of Hendrickson and Konnert. For the 4019 reflections between 10 and 3 A, with magnitudes at least 3 times their standard deviations, the R factor is 16.8%. The variation of the atomic temperature factors along the sequence indicates that the major flexibility regions are the amino acid and anticodon stems. The two strands of the amino acid helix exhibit large differential temperature factors, suggesting partial uncoiling or melting of the helix. In this work, the occupancy of all atoms was also varied. Residues D16 and D17 of the dihydrouridine loop as well as U33 and G37 of the anticodon loop have occupancies around 70%, indicating some local disorder or large-scale mobility at these positions. One hundred fifteen solvent molecules, including five magnesium ions, were found in difference maps. The role of several water molecules is clearly related to the stabilization of the secondary and tertiary interactions. The gold sites, which were not previously discussed, are described and show an energetically favored binding mode similar to that of cobalt and nickel complexes with nucleotides.     

Unit cell transormations in yeast phenylalanine transfer RNA crystals

The orthorhombic unit cell of crystalline yeast phenylalanine transfer RNA has dimensions $\text{a = 33}\text{A}\text{?}$, $\text{b = 56}\text{A}\text{?}$ and $\text{c = 161}\text{A}\text{?}$. When the mother liquor dries partially, a series of transformations takes place in which the a and b axes change very little but the c axis decreases abruptly first to 128 Å and then to 109 Å. In a closely related orthorhombic cell in a different space group the c axis is 104 Å. Although there is some loss in resolution in these smaller unit cells, the over-all distribution of scattering intensity does not change substantially. This suggests that the tRNA molecules can slide together along the c axis without a substantial change in internal structure.     

Yeast mitochondrial transfer RNA. Structure, coding properties and genome organization

The up-to-date data on mitochondrial tRNAs of yeast, their structures and peculiarities of these structures, anomalies of the mitochondrial genetic code and anticodons of tRNAs, the structure and number of tRNA genes are reviewed in the present paper. New information concerning 17 types of yeast mitochondrial tRNAs, deciphered by the authors of the paper are given; among them 8 types are first published. The likeness and differences of yeast mitochondrial tRNAs from their cytoplasmic counterparts are discussed by comparison with other organisms.     

Crystal structure of yeast phenylalanine transfer RNA. II. Structural features and functional implications.

The structural features of yeast phenylalanine transfer RNA are analyzed and documented in detail, based on atomic co-ordinates obtained from an extensive crystallographic refinement of the crystal structure of the molecule at 2.7 Å resolution (see preceding paper). We describe here: the relative orientation and the helicity of the base-paired stems; more definitive assignments of tertiary hydrogen bonds involving bases, riboses and phosphates; binding sites for magnesium hydrates, spermine and water; iriter-molecular contacts and base-stacking; flexibility of the molecule; conformational analysis of nucleotides in the structure. Among the more noteworthy features are a considerable irregularity in the helicity of the base-paired stems, a greater flexibility in the anticodon and aminoacyl acceptor arms, and a “coupling” among several conformational angles. The functional implications of these structural features are also discussed.