Optically detected magnetic resonance of the phosphorescent bases of Escherichia coli valine-specific transfer ribonucleic acid

Phosphorescence spectroscopy and optical detection of triplet state magnetic resonance (ODMR) spectroscopy have been used to characterize bases that contribute to the phosphorescence emission of Escherichia coli valine-specific transfer ribonucleic acid. When it is excited with 335-nm light, a short-lived phosphorescence with an origin near 435 nm is observed and is assigned to 4-thiouridine (s4U) at position 8 of the tRNA sequence. With excitation at 290-300 nm, a structured, long-lived phosphorescence is observed with an origin near 380 nm, in addition to the s4U phosphorescence. Comparison was made of the phosphorescence and ODMR spectra between Mg2+-containing and Mg2+-free tRNA samples. The s4U phosphorescence of the Mg2+-containing sample is more structured, and the peak is blue shifted relative to the Mg2+-free sample. Both samples give a single low-frequency (ca. 2.9 GHz) ODMR signal, but the high-frequency signal region (ca. 19-20 GHz) is structured. The Mg2+-containing sample has a partially resolved group of lines centered at 19.3 GHz, whereas the Mg2+-free sample has two broad bands centered at 19.2 and 20.0 gHz. The differences are attributed to effects of Mg2+ on the tRNA conformation. The ODMR signals observed by monitoring the long-lived phosphorescence are assigned to a pyrimidine nucleoside, possibly 5-(carboxy-methoxy)uridine in the anticodon.     

Assignment of the low field proton nuclear magnetic resonance spectrum of yeast phenylalanine transfer RNA to specific base pairs

High-resolution proton nuclear magnetic resonance spectra at 220 and 300 MHz have been used to investigate the base-pairing structure of fragments of yeast tRNA^Phe, of chemically modified tRNA^Phe and of intact tRNA^Phe. To a very good approximation the positions of the fragment spectra are additive within 0·2 part per million, indicating that factors responsible for certain structural features in the intact molecule are already present in the smaller fragments (half molecules, hairpins and $\text{3}\text{4}$ molecules). A simple first-order ring-current shift theory taken in conjunction with the cloverleaf model for tRNA^Phe (RajBhandary et al., 1967) has been used to predict the low-field (? 15 to ?11 part per million) nuclear magnetic resonance spectra and make assignments of the resolved resonances to ring NH protons of specific base pairs. The general agreement between the predicted and observed spectra to within 0·2 part per million confirms in detail the cloverleaf model for the secondary structure of tRNA^Phe in solution. It is also established that ring-current shifts are the principal factor responsible for the wide range of shifts observed in the low-field spectra. As a result it is evident that the resonances are very sensitive to small changes in the secondary structure and in some cases changes in the interbase distance as small as 0·2 Å could easily be detected. It is also clear from the analysis that certain of the resonances are sensitive to the tertiary structure of the molecule and specific examples are discussed. As with our previous study, we find no evidence for any strong Watson-Crick type base pairs beyond those predicted by the cloverleaf structure.     

Accessible and inaccessible bases in yeast phenylalanine transfer RNA as studied by chemical modification.

The selective modification of cytidine, uridine, guanosine and dihydrouridine residues in ³²P-labelled yeast phenylalanine transfer RNA has been studied by the use of specific reagents.The selective modification of cytidine residues with the reagent methoxyamine is described. Of the six cytidines in the single-stranded regions of the cloverleaf formula, only two are completely reactive, C74 and C75 at the 3′-terminus. Cm32 in the anticodon loop is reactive to only a small extent.The selective modifications of uridine and guanosine residues with 1-cyclohexyl 3-[2-morpholino(4)-ethyl] carbodiimide methotosylate, is described. The reagent is also shown to be reactive with dihydrouridine. In the single-stranded regions of the secondary structure of yeast phenylalanine transfer RNA there are 16 base residues which this reagent could be specific for. However, only G20, Gm34 and U47 are extensively modified, whilst U33 and D16 are partially modified. G18 is modified to a very small extent.The results obtained in this study are also in good agreement with previous chemical modification studied by other workers, carried out on unlabelled yeast phenylalanine transfer RNA using different reagents to the ones described here.The pattern of chemical modification is compared with the three-dimensional structure obtained by an X-ray crystallographic analysis of the same tRNA species. The correlation between exposed regions of the model and the regions of chemical reactivity are everywhere consistent.     

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.     

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.     

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.     

Study of triple-singlet energy transfer in an enzyme-dye complex using optical detection of magnetic resonance.

We have made optical detection of magnetic resonance (ODMR) measurements on the enzyme alpha-chymotrypsin, as well as on its complex with the dye, proflavin. Evidence that triplet-singlet energy transfer occurs in the complex is provided by the observation of characteristic tryptophan ODMR signals while monitoring the delayed fluorescence of the dye. The luminescence decay kinetics of the complex indicates that nontrivial triplet-singlet transfer originates from several (at least three) tryptophan residues of the enzyme. ODMR sensitivity can be enhanced by coupling the sublevels of a weakly radiative triplet state to a fluorescent dye which satisfies F?rster's (F?rster, T. (1948), Ann. Phys. (Leipzig) 2, 55; (1965), in Modern Quantum Chemistry, Istanbul Lectures, Part III, Sinanoglu, O., Ed., New York, N.Y., Academic Press, p 93) conditions for energy transfer.     

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.     

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.     

Nuclear magnetic resonance signal assignments of purified [13C]methyl-enriched yeast phenylalanine transfer ribonucleic acid

Yeast tRNA Phe, enriched in carbon-13 specifically at the naturally occurring methyl groups, has been produced through biosynthesis, then purified, and analyzed. Transfer RNA Phe was purified from the [13C]methyl-enriched, unfractionated tRNA that had been extracted from a methionine auxotroph of Saccharomyces cerevisiae [Agris, P. F., Kovacs, S. A. H., Smith, C., Kopper, R. H., & Schmidt, P. G. (1983) Biochemistry 22, 1402-1408]. The yeast had been grown in minimal medium supplemented with [13C]methylmethionine. Transfer RNA Phe purity and the full extent of nucleoside modification were confirmed by high-performance liquid chromatography of constituent nucleosides with simultaneous UV spectral identification and quantitation. Mass spectometry of [13C]methyl-enriched nucleosides and NMR of the tRNA indicated an enrichment of at least 70 atom %. Twelve resolved and prominent carbon-13 NMR signals from the tRNA were seen between 10 and 60 ppm. These have been assigned to 13 of the 14 naturally occurring methyl groups. However, the partially resolved signals assigned to the two 5-methylcytidines could not be assigned to their specific nucleoside positions of either 40 or 49 in the molecule. In addition, the partially resolved signals of the two methyl esters of wybutosine could not be distinguished. The methyl group found not to be enriched with 13C is bound to the ring carbon in the hypermodified nucleoside wybutosine (Y). A 13th enriched signal downfield (120.9 ppm) has been assigned to one of the two carbons added to guanosine to form the third ring in the biosynthesis of Y. The 13C enrichment of this ring carbon demonstrates its origin from the methionine methyl group.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Location of a platinum binding site in the structure of yeast phenylalanine transfer RNA

Trans-dichlorodiammineplatinum (II) reacts with yeast phenylalanine transfer RNA to yield a major platinum binding site. The tightly bound platinum has been located on the oligonucleotide Gm-A-A-Y-A-ψp containing the anticodon by standard fingerprinting methods using ³²P-labelled tRNA^Phe. This site corresponds to a single major platinum site identified during an X-ray crystallographic analysis of yeast tRNA^Phe. The solution studies have given confidence to the assignment of part of the 3 Å electron density map to the anticodon region of the molecular structure of yeast tRNA^Phe.     

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.     

High-resolution phosphorus nuclear magnetic resonance spectra of yeast phenylalanine transfer ribonucleic acid. Melting curves and relaxation effects.

In a continuation of our studies on structural effects on the 31P chemical shifts of nucleic acids, we present 31P NMR spectra of yeast phenylalanine tRNA in the presence and absence of Mg2+. Superconducting field (146 MHz) and 32-MHz 31P NMR spectra reveal approximately 15 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester. In the presence of 10 mM Mg2+, most scattered and main cluster signals do not shift between 22--66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles. At 70 degrees C, all of the signals merge into a single random coil conformation signal. Similar effects are observed in the absence of Mg2+ except that the transition melting temperature is approximately 20 degrees C lower. Measured spin-lattice and spin-spin relaxation times reveal another lower temperature transition besides the thermal denaturation process. A number of the scattered peaks are shifted (0.2--1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ as a result of this conformational transition between two intact tertiary structures. The loss of the scattered peaks in the absence of Mg2+ occurs in the temperature range expected for melting of a tertiary structure. An attempt to simulate the 31P spectra of tRNA Phe based upon the X-ray crystallographically determined phosphate ester torsional agles supports the suggestion that the large shifts in the scattered peaks are due to bond angle distortions in the tertiary structure.