Structure Of Yeast Phenylalanine Transfer RNA Research Articles

The Earliest Discovery of the Role of Magnesium Ions on Stabilizing the Tertiary Structure of the Transfer RNA and Its Biological Significance <br>—A Short Memoir

In early of 1960s, I was a graduate student studying on tRNA biochemistry. In the course of the research, the magnesium ions stabilized the tertiary structure of tRNA, resulting in its resistance to enzymatic degradation was discovered independently. The experiment of deaminated (denatured) tRNA obtained from native tRNA was designed and conducted and further proved the validity of this finding. It was found that magnesium ions could stabilize the tertiary structure of the natrive tRNA but could not stabilize structure of the deaminated tRNA. In term of the methodology, this stabilization technique has been widely applied in sequencing analysis of RNA and has greatly promoted the progress in the study of primary structure of RNA. More importantly, the stabilization of the tertiary structure of RNA by magnesium ions plays a key role both in the processing of messenger RNAs and the ribozyme activity. After our first article in Chinese was published in 1963, a paper of Nishimura & Novelli came into our note. The received date of their paper was March 22 of 1963, only 4 days earlier than that of our first paper. Thus, we and Nishimura & Novelli made almost at the same time the earliest discovery of the role of magnesium ions on stabilizing the tertiary structure of the transfer RNA and thus resulted in resistance of tRNA degradation by enzymes. However, this discovery was not initially appreciated for a period of time but was finally “visualized” and proved by X-ray crystal structure of yeast phenylalanine tRNA, which has provided more accurate information on the geometry of the magnesium-binding sites in tRNA.

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.

The crystal structure of yeast phenylalanine tRNA at 2.0 Å resolution: cleavage by Mg2+ in 15-year old crystals

We have re-determined the crystal structure of yeast tRNAPhe to 2.0 Å resolution using 15 year old crystals. The accuracy of the new structure, due both to higher resolution data and formerly unavailable refinement methods, consolidates the previous structural information, but also reveals novel details. In particular, the water structure around the tightly bound Mg2+ is now clearly resolved, and hence provides more accurate information on the geometry of the magnesium-binding sites and the role of water molecules in coordinating the metal ions to the tRNA. We have assigned a total of ten magnesium ions and identified a partly conserved geometry for high-affinity Mg2+ binding. In the electron density map there is also clear density for a spermine molecule binding in the major groove of the TΨC arm and also contacting a symmetry-related tRNA molecule. Interestingly, we have also found that two specific regions of the tRNA in the crystals are partially cleaved. The sites of hydrolysis are within the D and anticodon loops in the vicinity of Mg2+.

A model for the tertiary structure of mammalian mitochondrial transfer RNAs lacking the entire 'dihydrouridine' loop and stem.

The mammalian mitochondrial tRNA(AGY)Ser is unique in lacking the entire dihydrouridine arm. This reduces its secondary structure to a 'truncated cloverleaf'. Experimental evidence on the tertiary structure has been obtained by chemically probing the conformation of both the bovine and human species in their native conformation and at various stages of denaturation. A structural model of the bovine tRNA is presented based on the results of this chemical probing, on a comparison between nine homologous 'truncated cloverleaf' secondary structures and on analogies with the crystal structure of yeast phenylalanine tRNA. The proposed structure is very similar in shape to that of yeast tRNA(Phe) but is slightly smaller in size. It is defined by a unique set of tertiary interactions. Structural considerations suggest that other mammalian mitochondrial tRNAs have smaller dimensions as well.

Nuclear Overhauser assignment of the imino protons of the acceptor helix and the ribothymidine helix in the nuclear magnetic resonance spectrum of Escherichia coli isoleucine transfer ribonucleic acid: evidence for costacked helices in solution.

In a previous study we showed that, in the low-field nuclear magnetic resonance (NMR) spectrum of Escherichia coli tRNAVal1, the hydrogen-bonded imino protons of the four Watson-Crick base pairs in the dihydrouridine helix could be assigned on the basis of their proximity to the imino proton of s4U8 by means of sequential Nuclear Overhauser (NOE) connectivity (Hare & Reid, 1982). In the present paper we have used the nearest-neighbor NOE technique to assign all the imino proton resonances of the acceptor helix and the ribothymidine helix of E. coli tRNAIle1. As reference points we used the GU-type base pairs located at positions 5 and 49 in this molecule which are readily identifiable in the NMR spectrum by virtue of containing two imino protons in the same base pair. From UG5 the imino protons of base pairs 4,3,2,1 and 6,7 could be assigned by through-space NOE connectivity. Similarly the imino protons of 50,51,52,53 were assigned by their spatial relationship to G psi 49. NOE connectivity also revealed a base pair stacked on the external side of GC53 which, by analogy with the crystal structure of yeast phenylalanine tRNA, is presumed to be the tertiary pair T54-A58. This was confirmed by NOE connectivity from the thymine methyl resonance. In addition to assigning 17 of the imino resonances in the low-field NMR spectrum of isoleucine tRNA, these NOE studies show that the acceptor helix and the ribothymidine helix are stacked on each other in solution in that base pairs 7 and 49 are directly connected in space.

Chemical modification analysis of ion-dependent changes in the solution structure of yeast phenylalanine tRNA

Chemical modification analysis of ion-dependent changes in the solution structure of yeast phenylalanine tRNA

Stabilization of the tertiary structure of yeast phenylalanine tRNA by [Co(NH 3) 6] 3+: X-ray evidence for hydrogen bonding to pairs of guanine bases in the major groove

The sites of three [Co(NH 3) 6] 3+ ions bound to the phenylalanine tRNA of yeast have been determined by X-ray diffraction analysis. [Co(NH 3) 6] 3+ binds to purine-purine sequences in yeast tRNA Phe. It is different from the binding of Co 2+, which binds to the base and phosphate of residue G15. There are no direct metal-nucleotide bonds, although hydrogen bonding of the coordinated ammines to double-helical guanylguanosine sequences in the major groove and to phosphate oxygens in neighboring polynucleotide strands increases the stability of the structure. Hydrogen-bonding appears to be via cis ammine ligands to N(7) and O(6) positions of adjacent purine bases.

Changes in the solution structure of yeast phenylalanine transfer ribonucleic acid associated with aminoacylation and magnesium binding.

The effect of aminoacylation on the structure of yeast phenylalanine tRNA was evaluated by laser light scattering. In these experiments, the translational diffusion coefficient (D20,w) of phenylalanyl-tRNA was monitored continuously during spontaneous deacylation in a variety of solution conditions. The results reveal that significant changes can occur in the hydrodynamic volume and electric charge as a consequence of aminoacylation but that the effects are magnesium dependent. At neutral pH, 20 degrees C, and 0.1 M salt, the D20,w value increased by 18% when deacylation occurred in 2--10 mM Mg2+ concentrations while no change in diffusivity was observed for tRNA deacylating in 0.5--1.0 mM Mg2+. The Mg2+ concentration dependence of the D20,w changes behaves in highly cooperative manner. The electric charges of aminoacyl-tRNA and nonacylated tRNA in 1 and 10 mM Mg2+ were estimated from the diffusive virial coefficients. In the higher Mg2+ conditions, aminoacyl-tRNA has a charge of 15 +/- 2e- while that of the nonacylated form is 10 +/- 2e-; both acylated and nonacylated tRNA have a charge of 11 +/- 4e- in 1 mM Mg2+. Taken together, the results indicate that aminoacylation permits the binding of additional Mg2+, resulting, in turn, in the formation of a more extended conformer of lower diffusivity and greater negative charge. The results also provide a possible explanation for several contradictory results in the literature.

Intercalation conformations in single- and double-stranded nucleic acids

Two regions in the crystal structure of yeast phenylalanine tRNA, where single-stranded loops interact by intercalation, have been examined in detail. There are four examples of a nucleotide base from one loop intercalating between two sequential bases of another loop in these two regions. These four dinucleoside phosphate conformations serve as models for intercalation in single-stranded nucleic acids. Double-stranded DNA and RNA polymers were constructed by computer model building methods, which incorporated the dinucleoside phosphate conformations found in these single-stranded, intercalation regions in otherwise standard double-helices. The results suggest that it is unlikely that there is a unique intercalation geometry for either single- or double-stranded nucleic acids, but that nucleic acids may assume one of a variety of intercalation geometries which will best accommodate a particular intercalating agent for a particular base sequence.

Crystal structure of yeast phenylalanine transfer RNA: I. Crystallographic refinement

We present the results of the final stage of the X-ray Crystallographic studies of yeast phenylalanine transfer RNA in an orthorhombic crystal form. The crystal structure of the transfer RNA has been refined by a least-squares procedure to minimize the difference between the observed ( F o) and calculated ( F c) structure factors from X-ray diffraction patterns. The final Crystallographic discrepancy index, R = ∑¦F o −F c ¦/∑F o , is 0.198, based upon 8426 structure factors with magnitudes over twice the estimated standard deviation, corresponding to 96.4% of the complete set of data with resolutions up to 2.7 Å. During the refinement, bond lengths and angles within each phosphate group and each nucleoside (base plus sugar) were constrained exactly to their appropriate standard values, while those for the linkages between the nucleosides and phosphates were elastically restrained close to their standard values. The details of the application of the constraint-restraint least-squares (CORELS) refinement method to the crystal structure of yeast phenylalanine tRNA are described in this paper. A complete list of atomic co-ordinates and the rigid group thermal factors are presented. The stereochemical details of this structure and their functional implications are described in the following paper.

A structure-factor least-squares refinement procedure for macromolecular structures using constrainedandrestrained parameters

A method of structure-factor least-squares refinement of constrained groups linked by distance restraints has been developed for the refinement of macromolecular structures. Each constrained group can have any number of variable dihedral rotation parameters within the group in addition to the rigid-body translational, rotational and thermal parameters. The matrix of normal equations may be either full or sparse and provision is made for solution by matrix inversion or the conjugate-gradient iterative method. This procedure has been successfully used for 3 Å data and should be applicable even for lower-resolution data and especially for cases with a poorer data-per-atom ratio. The structure of yeast phenylalanine tRNA has been refined with this procedure from a starting crystallographic R value of 42% to a final R value of 25% with isotropic 'group" thermal parameters and 22% with isotropic atomic thermal parameters for 8207 independent reflections at 2.7 Å resolution. The proper stereochemistry of bond distances, angles and van der Waals contacts for the restrained atoms was maintained within reasonable limits throughout the refinement. Although originally developed for nucleic acids, this procedure is directly applicable to the refinement of protein structures. In addition, a combination of applying distance restraints between groups and least-squares fitting of these groups to target coordinates has been used purely as an idealization process for imposing proper stereochemistry on an approximate model.

The general structure of transfer RNA molecules.

The three-dimensional structure of yeast phenylalanine tRNA serves as a useful basis for understanding the tertiary structure of all tRNAs. A large number of tRNA sequences have been surveyed and some general conclusions are drawn. There are only a few regions in the molecule in which there are differences in the number of nucleotides; and the structure of yeast phenylalanine tRNA can accommodate these differences by forming or enlarging protuberances on the surface of the basic framework molecule. The nature and distribution of the differences in number of nucleotides are surveyed and possible hydrogen bonding interactions are discussed for a number of tRNA classes. The two most significant features of the molecule are the large number of stacking interactions which are seen to include most of the nucleotides in the molecule and the system of specific hydrogen bonding interactions. It is likely that these stabilizing elements are preserved in all tRNA structures.

Structure of yeast phenylalanine tRNA at 3 A resolution.

The structure of a tRNA has been determined by isomorphous replacement. Some of the interactions which maintain the tertiary structure are of a novel type. Our model differs significantly from one which has recently been proposed.