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.     

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.     

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.     

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.     

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.     

Internal motions in yeast phenylalanine transfer RNA from 13C NMR relaxation rates of modified base methyl groups: a model-free approach

Internal motions at specific locations through yeast phenylalanine tRNA were measured by using nucleic acid biosynthetically enriched in 13C at modified base methyl groups. Carbon NMR spectra of isotopically enriched tRNA(Phe) reveal 12 individual peaks for 13 of the 14 methyl groups known to be present. The two methyls of N2,N2-dimethylguanosine (m22G-26) have indistinguishable resonances, whereas the fourteenth methyl bound to ring carbon-11 of the hypermodified nucleoside 3' adjacent to the anticodon, wyosine (Y-37), does not come from the [methyl-13C]methionine substrate. Assignments to individual nucleosides within the tRNA were made on the basis of chemical shifts of the mononucleosides [Agris, P. F., Kovacs, S. A. H., Smith, C., Kopper, R. A., & Schmidt, P. G. (1983) Biochemistry 22, 1402-1408; Smith, C., Schmidt, P. G., Petsch, J., & Agris, P. F. (1985) Biochemistry 24, 1434-1440] and correlation of 13C resonances with proton NMR chemical shifts via two-dimensional heteronuclear proton-carbon correlation spectroscopy [Agris, P. F., Sierzputowska-Gracz, H., & Smith, C. (1986) Biochemistry 25, 5126-5131]. Values of 13C longitudinal relaxation (T1) and the nuclear Overhauser enhancements (NOE) were determined at 22.5, 75.5, and 118 MHz for tRNA(Phe) in a physiological buffer solution with 10 mM MgCl2, at 22 degrees C. These data were used to extract two physical parameters that define the system with regard to fast internal motion: the generalized order parameters (S2) and effective correlation times (tau e) for internal motion of the C-H internuclear vectors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientation of double-helical segments in crystals of yeast phenylalanine transfer RNA

A search of the X-ray intensities of the P2₁ crystal form of yeast transfer RNA^Phe has revealed the orientation of the double-helical segments in the crystal. Because of the ambiguity imposed by the crystal symmetry on choosing the helices belonging to the same molecule, and because of the difficulty of determining lengths and positions of helices, a unique model cannot be deduced, but only a small number of types are possible. Among the possibilities are a “boot”-shaped molecule, which may be derived from an earlier model proposed by the author, by bending out the anticodon arm, and also an L-shaped molecule. The latter is, however, not oriented in the way proposed by Kim et al. (1973) for the case of the closely related orthorhombic crystal form.     

Functional mutants of phenylalanine transfer RNA from Escherichia coli.

The gene pheV from Escherichia coli, coding for tRNAPhe and carried on a plasmid, has been mutagenised with hydroxylamine. Mutants in the structural gene have been identified using two criteria: (i) de-attenuation of beta-galactosidase expression, while under the control of the attenuator region of the pheS,T operon by means of an operon fusion; (ii) loss of ability to complement thermosensitivity of a mutant Phe-tRNA synthetase. Mutants showing de-attenuation were sequenced and two nucleotide changes identified: G44----A44 (found five times) and m7G46----A46 (found once). Sequencing of mutants that lost complementation identified two further tRNA mutants, C2---U2 and G15----A15; the mutant m7G46----A46 was also re-isolated by this criterion. Three of the mutants involve bases implicated in tertiary rather than secondary structure hydrogen bonding. One hypothesis for the mechanism of de-attenuation is that mutant tRNAPhe molecules compete with the wild-type tRNAPhe on the ribosome but are inefficient at some step in the elongation process.     

Correlation between chemical modification and surface accessibility in yeast phenylalanine transfer RNA

Chemical reactivities of the functional groups of yeast phenylalanine transfer RNA are compared with surface accessibilities of the groups calculated with various probe radii representing effective radii of the chemical reagents used. We observe 97% agreement with the hypothesis that the chemically modified bases are those with the greatest surface accessibility. This overall strong correlation supports the conclusion that base exposure in an important determinant of chemical modification in this polynucleotide.     

The selective iodination of yeast phenylalanine transfer RNA with 125I

Yeast tRNA^Phe has been labelled with ¹²⁵I under conditions which conserve the tertiary structure. Significant labelling was only found to occur on specific cytidines in single stranded regions, while other cytidines in single stranded regions and all those in the double stranded region underwent iodination to a very small extent. The pattern obtained from iodine labelling satisfies the conformation of a model recently proposed for this tRNA.     

X-ray crystallographic studies of polymorphic forms of yeast phenylalanine transfer RNA

Yeast phenylalanine transfer RNA has been found to crystallize in five different crystal systems involving eight different space groups. The X-ray diffraction characteristics of these forms are described. One of the orthorhombic forms yields a diffraction pattern with higher resolution than either the hexagonal, the cubic or the monoclinic forms. One region of this orthorhombic diffraction pattern is particularly sensitive to X-ray exposure and to changes in the concentration of various solutes. The diffraction pattern from the cubic crystal form extends to a resolution of 3 Å, and there are a number of strong reflections in the 3 to 4 Å region which suggest that double-helical segments of the tRNA molecules are oriented along the 4-fold axes. Some comments are made regarding the nature of the polymorphism in the transfer RNA crystals.