Structure Of Yeast tRNAPhe Research Articles

The crystal structure of yeast phenylalanine tRNA at 1.93 A resolution: a classic structure revisited.

The crystal structure of the monoclinic form of yeast phenylalanine tRNA has been redetermined at a resolution of 1.93 A. The structure of yeast tRNAphe described here is more accurate than its predecessors not only because it incorporates higher resolution data, but also because it has been refined using techniques that had not been developed when its predecessors were determined more than 20 years ago. The 1.93 A resolution version of this structure differs interestingly from its predecessors in its details. In loop regions particularly, the backbone torsion angles in the new structure are not the same as those reported earlier. Several new divalent cation binding sites have been identified, and the water structure that has emerged is also different.

Ternary complex between elongation factor Tu•GTP and Phe-tRNA Phe

The effect of aminoacylation and ternary complex formation with elongation factor Tu•GTP on the tertiary structure of yeast tRNA Phe was examined by 1H-NMR spectroscopy. Esterification of phenylalanine to tRNA Phe does not lead to changes with respect to the secondary and tertiary base pair interactions of tRNA. Complex formation of Phe-tRNA Phe with elongation factor Tu•GTP results in a broadening of all imino proton resonances of the tRNA. The chemical shifts of several NH proton resonances are slightly changed as compared to free tRNA, indicating a minor conformational rearrangement of Phe-tRNA Phe upon binding to elongation factor Tu•GTP. All NH proton resonances corresponding to the secondary and tertiary base pairs of tRNA, except those arising from the first three base pairs in the aminoacyl stem, are detectable in the Phe-tRNA Phe•elongation factor Tu•GTP ternary complex. Thus, although the interactions between elongation factor Tu and tRNA accelerate the rate of NH proton exchange in the aminoacyl stem-region, the Phe-tRNA Phe preserves its typical L-shaped tertiary structure in the complex. At high (> 10 −4 M) ligand concentrations a complex between tRNA Phe and elongation factor Tu•GDP can be detected on the NMR time-scale. Formation of this complex is inhibited by the presence of any RNA not related to the tRNA structure. Using the known tertiary structures of yeast tRNA Phe and Thermus thermophilus elongation factor Tu in its active, GTP form, a model of the ternary complex was constructed.

Loop stereochemistry and dynamics in transfer RNA.

The stereochemistry and the dynamics of two loops of yeast tRNA-asp, the thymine loop and the anticodon loop, are compared in the hope of a better understanding of the relationships between loop sequence and loop topology. Both loops are seven residues long and both present sharp turns after the second residue, U33 and psi 55, stabilized by hydrogen bonds between N3-H of the pyrimidine and the phosphates of C36 and A58 and stacking interactions of the pyrimidine ring with the phosphates of U35 and A57, respectively. In the thymine loop, the two purines following C56, A57 and A58, open up to leave space for the intercalation of the first invariant guanine residue of the D-loop, while the two pyrimidine bases, which follow A58, turn away from the stacking pattern of the thymine arm and stack instead with the last base pair of the dihydrouridine arm A15-U48. In the anticodon loop, however, the bases G34 to C38 form an helical stack in continuity with the anticodon stem on the 3'-end. At the same time C36 forms Watson-Crick hydrogen bonds with G34 of a twofold symmetrically related molecule. The anticodon-anticodon base pairing interactions between symmetrically-related molecules are stabilized by stacking with the modified base G37 on both sides of the triplet. Some comparisons are made with the structure of yeast tRNA-phe and some implications about the structure of mitochondrial tRNAs are discussed.

Hydrogen Bond Indices and Tertiary Structure of Yeast tRNAPhe

The rigidity and stability of the tertiary structure of yeast tRNAPhe is related to a bond index obtained in an IEHT (iterative extended Hückel theory) calculation. The index permits a quantitative estimate of the electron density along the hydrogen bond, having thus an appealing physical meaning. The results indicate that Hoogsteen-type bonds have, as expected, greater electronic population than Watson-Crick type ones. Other non-Watson-Crick pairs, the wobble pair and G15-C48, exhibit high values of the index for the NH...O bond. In the triples, the electron density of the hydrogen bridges does not weaken (compared with the one of the pairs involved). Contour density maps are shown and dipolar moments of pairs and triples are qualitatively discussed.

The tertiary structure of yeast tRNA Phe in solution studied by phosphodiester bond modification with ethylnitrosourea

Chemical modification of tRNAs with base-specific reagents was successfully used for investigation of their structure in solution f 11. Using base-specific reagents allows the identi~cation of bases involved in hydrogen bond formation in tRNA structure. The development of a chemical modification method that would provide more information could be the use of reagents able to modify the riboses or the phosphates common to all nucleotide residues in tRNA. Studying the reactivity of these residues scattered throughout the entire tRNA molecule would then provide information about the backbone interactions in the tRNA and about the folding of the tRNA polynucleotide chain. We present here results of chemical modi~~t~on of yeast tRNAPhe with ethylnitrosourea, a reagent alkylating phosphodiester bonds in nucleic acids [2,3]. In the unfolded tRNAPhe all phosphates were reactive toward this reagent. In contrast in the folded tRNAPhe phosphates in different sites of the molecule were remarkably different in their reactivities toward the reagent. Phosphates in the exposed helical regions of tRNAphe(for instance in the anticodon arm) readily react with ethylnitrosourea, whilst the reactivity of some phosphates in the regions known to be involved in the tertiary structure formation is strongly reduced. The most pronouced effects were observed for the phosphates in positions 9,10,11,19,49,58,59 and 60.

Sequential folding of transfer RNA: A nuclear magnetic resonance study of successively longer tRNA fragments with a common 5′ end

Most folding studies on proteins and nucleic acids have been addressed to the transition between the folded and unfolded states of an intact molecule, where an entire residue sequence is present during the folding event. However, since these polymers are synthesized sequentially from one terminus to the other in vivo, their folding pathways may be influenced greatly by the sequential appearance of the residues as a function of time. The three-dimensional structure of yeast tRNA Phe in the crystalline state is correlated with 360 MHz proton nuclear magnetic resonances from three fragments plus an intact molecule of the tRNA that share a common 5′ end and are in a solution condition similar to that of the crystal structure. This has allowed identification of folded structures present in the fragments and presumably present in the growing tRNA molecule as it is being synthesized from the 5′ end. The experiments show that only the correct stems are formed in the fragments; no additional or competing helical region is produced. This suggests that in the biosynthesis of this tRNA, correct folding of helical stems occurs before the entire molecule is formed. Further, some of the tertiary interactions (hydrogen bonds) found in the crystal structure are also probably present before the synthesis is completed. These findings are generalized to consider the precursor of the tRNA as well as other tRNAs.

Further refinement of the structure of yeast tRNA Phe

We have refined the monoclinic crystal structure of yeast tRNA Phe against a complete set of X-ray data at 2.5 Å resolution, using real-space refinement and a combination of energy minimisation and crystallographic least-squares. This refinement has allowed us to define the conformation of residue D16, and to make corrections to Y37 and A76. We have found an additional magnesium binding site (making a total of four), a number of water molecules, and a possible spermine molecule. A revised list of torsion angles is given.

Effect of magnesium and polyamines on the structure of yeast tRNA Phe

The effect of magnesium and polyamines (spermine, spermidine, putrescine and cadaverine) on the structure of yeast tRNA Phe has been investigated. It is found that magnesium induces structural changes and stabilizes hydrogen bonds in the temperature range 22–44°C in 0.17 M sodium. The number of Mg 2+ which affect tRNA structure increases from 1 ± 1 at 22°C to 4 ± 1 at 44°C and the number of additional base pairs formed in the presence of magnesium increases from 1 ± 1 at 22°C to 4 ± 1 at 44°C. The spectral changes are more-or-less sequential. The polyamine spermine stabilizes some, but not all, of the structural features stabilized by magnesium at 44°C, and the combination of magnesium and spermine, at low levels, is more effective than either cation alone in stabilizing tRNA structure. Comparison of the effects of spermine, spermidine, putrescine and cadaverine indicates that it is the asymmetric triamine unit which is important in the stabilization. Some spectral changes induced by magnesium can be assigned to stabilization of specific tertiary structure interactions and to alteration of stacking adjacent to U 8 · A 14.

Demonstration of a tertiary interaction in solution between the extra arm and the D-stem in two different transfer RNA's by NMR

According to the X-ray structure of yeast tRNAPhe at 2.5 A resolution, a hydrogen bond is formed between m7G46 and G22. By removal of this m7G46-residue we demonstrate that this interaction is present in solution as well. Comparison of the 1H 360 MHz NMR spectra of intact yeast tRNAPhe and its m7G-excised derivative locates the position of this tertiary H-bond at 12.5 ppm downfield from DSS. Additional evidence for the presence of this interaction in solution comes from a comparison of 1H NMR spectra of E. coli tRNAf1Met and E. coli tRNAf3Met, which differ only in a single position in the extra arm. In tRNAf3Met residue 47 is a m7G-residue, whereas in tRNAf3Met it is A, resulting in the absence of the m7G47 - G23 - C13 triple interaction, characteristic of tRNAf1Met. The resonance position of this tertiary interaction in tRNAf1Met is located around -13.6 ppm, a chemical shift difference of 1.1 ppm with respect to the position observed for tRNAPhe. The origin of this chemical shift difference is discussed in relation to the structure of their respective augmented D-helices.

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.

On the structure of yeast tRNA Phe . Complementary-oligonucleotide binding studies.

The availability of (yeast)tRNAPhe for helix formation with complementary oligoribonucleotides has been explored by equilibrium dialysis studies. Oligoribonucleotides complementary to sequences which are double‐stranded, according to the cloverleaf model of tRNAPhe, did not bind. Two regions of tRNAPhe were fully acessible to oligonucleotide binding: (a) the 3′‐terminus; (b) the sequence (3′‐5′)U‐G‐A‐A‐Y (33–37) of the anticodon loop. Two other regions were accessible to oligonucleotide binding: (a) part of the extra arm; (b) the (3′‐5′)T‐Ψ‐C sequence of the ribosylthymine loop. However, the binding constants of trimers and tetramers complementary to these parts of tRNAPhe are very low.The data indicate that the anticodon loop of tRNAPhe has an asymmetric structure, which is not compatible with the Fuller‐Hodgson model. The low binding constants for oligonucleotides complementary to the extra arm or to the TpΨpC sequence are discussed. They seem to be due either to sterical hindrance or to different conformations of tRNA. In the latter case, one conformation would be accessible to complementary oligonucleotides for helix formation, the other would not. Present available oligonucleotide binding data of four tRNAs suggest that each tRNA has an individual pairing scheme, according to which the loops of the cloverleaf contribute to the three‐dimensional structure.