i. INTRODUCTION

ii. BACKGROUND: Historical & Experimental

iii. OVERALL STRUCTURE

iv. SECONDARY STRUCTURE

v. TERTIARY STRUCTURE: Base-base Interaction, Base-backbone Interaction, Backbone-backbone Interaction, Metal-tRNA Interaction, Spermine-tRNA interaction & Water-tRNA interaction.

vi. CONFORMATION OF THE ANTICODON LOOP

vii. INTERMOLECULAR CONTACTS

viii. INTERCALATION GEOMETRY

ix. FUNCTIONAL IMPLICATIONS OF THE THREE-DIMENSIONAL STRUCTURE: Molecular Design, Structural Stability and Specificity, Flexibility of the Molecule, Modified Nucleosides & T-Y-C Corner.

INTRODUCTION. Transfer RNA is responsible for numerous biological function. tRNA is involved in the central role of protein synthesis, the regulation of enzyme biosynthesis, the synthesis of cell wall, and serve as primers to RNA-dependent polymerase in reverse transcription among other many functions. The overall three dimensional conformation of tRNA is a major contributor to its multi-functionality.

BACKGROUND: Historical. Following the advent of refined techniques in isolation and sequencing mRNAs in the 1960s, the deciphering of the genetic code dictionary was just a matter of time. Within the first year following the breakthrough (1961), Marshall Nirenberg and Heinrich Matthaei established that -UUU- condon specified Phe. In Kim's Phe_tRNA structure (1978), the codon triplet that specifies Phenylalanine is -GAA-.

BACKGROUND: Experiment. The structure of yeast (Saccharomyces Cerevisiae) phenylalanine transfer RNA (sometimes abbreviated Phe_tRNA) is depicted on the following Chime/RasMolŠ exhibit. The overall structure of Phe_tRNA interpreted from x-ray diffraction analysis at a 2.7 Angstroms resolution by Sung-Hou Kim and colleagues resembles the shape of the letter "L" <Click to view L shape tRNA>. The PDB reference code for the yeast is 6TNA, published in Nov 16, 1978. During the refinement of this model, bond lengths and angles within each phosphate group and each nucleoside were constricted, while those for the linkages between the nucleosides and phosphates were restrained to standard values.

OVERALL STRUCTURE. The polynucleotide backbone of Phe_tRNA folds to form one continuous double-helical arm with the acceptor stem and the T stem (Red) <Click to view 1st double-helical arm>. A second double-helical arm is formed by D stem and the anticodon stem (Orange) <2nd double-helical arm>. Each of these extensions is roughly 60 Angstroms in length and about 20 Angstroms in diameter, while the distance between the its two ends is 80 Angstroms. Each stem is a right-handed double helix running antiparallel to each other. The site of peptide elongation is located at one extreme, the 3'-end (3'-OH of A76 nucleotide) <Click to view 3'-OH of A76 nucleotide>. At the other extreme, the anticodon stem is responsible for codon recognition on the messenger RNA. The T loop, located at the corner of the "L," is believed to interact with ribosomal RNA. The overall structure of tRNA minimizes interference among its functional regions by isolating its functionally important sites.

SECONDARY STRUCTURE. tRNA can be subdivided into four regions: (1) the amino acid stem, where the amino acid residue is carried by its 3'-OH terminal <Click button to view amino acid stem (Red)> <Show 3'-OH end (Magneta)>; (2) the D stem which frequently contains the modified base dihydrouridine <Click button to view D stem (Blue)>; (3) the anticodon stem which contains the anticodon triplet of bases that complementary to the codon of the mRNA <Click to button view anticodon stem (Green)>; (4) T stem containing the pseudoridine base <Click button to view T stem (Yellow)>; and (5) the variable arm which is the site of greatest variability in RNA <Click button to view variable stem (White)>. All four helixes have shallow and deep grooves, while the nucleotide residues are tilted away from its helical axis. Moreover the ribose sugar residues are in a 3'-endo conformation

All four stems have high irregularity in their helical parameters (Sussman & Kim, 1978) evidenced by the large standard deviation associated with their helicity (irregularity surpasses the range of error of 0.1A based on the evidence of a previous Sussman & Kim publication). Kim, Sussman and colleagues concluded the irregularities to be real.

All base-pairs but G4-U69 present in the stem are Watson-Crick types <Click to view G4-U69 base-pair>.

TERTIARY STRUCTURE: Base-base Interaction. Several factors contribute to the stability of the tertiary structure of tRNA. all contribute to the stability of tRNA. Base-base interaction are based on non Watson-Crick tertiary hydrogen bonding between bases with the exception of

Several factors contribute to the stability of the tertiary structure of tRNA. Hydrogen bonding and stacking between base pairs, hydrogen bonding between base and backbone, between backbone and backbone, binding of metal ions, spermines and water interactions all contribute to the stability of tRNA. Base-base interaction are based on non Watson-Crick tertiary hydrogen bonding between bases with the exception of G19-C56 (Red) <Click to show G19-C56 (Red) <Click to show G19-C56 non Watson-Crick base-pair> . Here shown are nine tertiary base-pairs <Click button to view 9 tertiary base-pairs>. Notice the stacking of the bases from the three dimensional structure with the exception of five bases (D16, D17, G20, U47, and A76) <Click to view non stacking base-pairs (White)>.

TERTIARY STRUCTURE: Base-backbone Interaction. The assignment of tertiary hydrogen bonding between base and backbone involves the assignment of 12 hydrogen bonds based on the criteria of distance and stereogeometry. Five of the 12 hydrogen bonds involve the O2' hydroxyl groups from the sugar.

TERTIARY STRUCTURE: Backbone-backbone Interaction. Distance and stereogeometry were again the criteria used to assign hydrogen bonds within the backbone. All hydrogen bonds under this category involve O2' hydroxyl groups except for cases where one could assign hydrogen bonds between O5' and purine C8 or pyrimidine C6 of the same nucleotide. These CH----O hydrogen bonds are similar to the dinucleoside phosphates UpA and ApU (Sussman, 1972; Rubin, 1972; Seeman, 1976). The prevalence of O2' hydroxyl group H-bonds could be the reason behind RNA's structural nucleic acids functions as found in tRNAs and ribosomal RNAs. Similarly this also excludes the participation of DNAs in these processes.

TERTIARY STRUCTURE: Metal-tRNA Interaction. Specific functionally conformation in tRNAs is achieved via site-specific bound magnesium ions (Fresco, 1966). There are essentially four sites where magnesium hydrates are bound to tRNA. Two are located at the D loop (Blue) <Click to view D loop>, one in the anticodon loop (Green) <Click to view Anticodon Stem>, and the last one in the between the region formed by residues 8 through 12 (Magneta) <Click to view residue 8-12 magnesium binding site>. Magnesium metals thus are major contributors to the in stability of tRNA's loops and sharp turns.

TERTIARY STRUCTURE: Spermine-tRNA Interaction. Polyamines like and also play an important role in stabilizing tRNA's tertiary conformation (Saikat & Cohen, 1976). Kim and his colleagues used spermine in the crystallization Phe_tRNA. They found out two possible sites for spermine binding: the formed by the and the The second candidate site is formed by the and the .

Polyamines like spermine and spermidines also play an important role in stabilizing tRNA's tertiary conformation (Saikat & Cohen, 1976). Kim and his colleagues used spermine in the crystallization Phe_tRNA. They found out two possible sites for spermine binding: the deep groove formed by the T stem (Yellow) <Click to view T stem> and the acceptor arm <View Acceptor stem>. The D stem <Click to view D loop> and the anticodon arm (Green) <Click to view Anticodon Stem> forms the second candidate site.

TERTIARY STRUCTURE: Water-tRNA Interaction. There are about 60 molecules bound to tRNA. Because of weak electron densities from these water molecules, the number of molecules involved in stabilization must be taken with reservation.

Overall the three-dimensional structure of tRNA is stabilized by the sum of all the above interactions. Base-stacking and base-pairing between the nucleotides primarily stabilize the arms of the "L". At the junction of the arms of "L," base-stacking combined with base-base, base-backbone, and backbone-backbone hydrogen bonding help stabilize the sharp turn. Moreover, three of the four stabilizing magnesium ions are located at this central region to further stabilize the loops and turns. The remaining magnesium ion is found at the anticodon stem, presumably stabilizing this functionally essential region <Click to view the stabilization due to base stacking>.

CONFORMATION OF THE ANTICONDON LOOP. Unlike the rest of the molecule, hydrogen bonding does not stabilize tRNA's anticodon arm

INTERMOLECULAR CONTACTS. The tRNA crystal is packed in orthorhombic crystalline form, occupying 20-25% of the lattice's volume. There are basically four small regions of contact in tRNA. First, an extensive intermolecular contact occurs at the D loop. Residues 17, 19 and 20 of one molecule <Click button to display residues 17, 19 and 20> are in contact with residues residues 14, 15 and 16 of a second molecule <Click to show residue 14, 15 and 16>, and residue 56 of a third molecule <Click to show residue 14, 15 and 16>. These molecule contacts are primarily van der Waals forces. Two intermolecular hydrogen bonds between O2' of one residue and the phosphate oxygen of a second molecule's residue 20, and between O2' of C13 and O4 of D17. Two hydrated Mg2+ further stabilizes this region.

Second, T stem of one molecule contacts with the acceptor stem of a second molecule forming a 14° angle. This contact involves (1) one hydrogen bond between the O2' hydroxyl group of ribose 5 of one and the phosphate oxygen of the other's residue 53; (2) and one van der Waals interaction.

The third region of contact occurs at the anticodon loop's extremity . Residue 34's base of one molecule stack with same residue of a second molecule .

The fourth molecular contact occurs at the 3' end of tRNA. Residue 76 of the first contacts residue 37's modified base of the second. Residues 75 and 76 of the first make contact with residues 28, 29 and 43 of a third molecule. Seven total intermolecular hydrogen bonds and seven total van der Waals' contacts can be made in this region.

INTERCALATION GEOMETRY. tRNA displays four regions of two consecutive bases intercalated with a third base. Two of these intercalating regions occur where the T and D stems contact. The base stacking sequence G19-G57-G18-A58 is generated by the intercalation of two bases from residues 58 and 57 with two other bases from residues 18 and 19. The third involves residues 45 and 46 of the variable loop intercalated with the Adenine 9 (G18-A9-G19). The fourth example of intercalating bases involves residues 8 and 9 and the base triplet C13-G22-G46. The 3' end ribose assumes a C2'-endo puckering (i.e. G18-G19).

FUNCTIONAL IMPLICATIONS OF THE THREE-DIMENSIONAL STRUCTURES: Molecular design. The 3' end, site of amino acid attachment or peptide growth, is separated from the anticodon triplet by 80A. These two functional region are about 60A away from the T-Y-C conserved sequence, which is involved in recognition between tRNA and ribosomal RNA. Since all three functionally important regions may interact with ribosomal RNA and other RNAs simultaneously it is important that they are physically maximally separate from each other.

FUNCTIONAL IMPLICATIONS OF THE THREE-DIMENSIONAL STRUCTURES: Structural stability and specificity. tRNA is a very stable molecule that can easily renature. The high degree stability is essential for the multi-functionality of tRNA. are stacked indicating is primarily a stabilizing factor. Further more, specifies the overall structural framework of tRNA. These base-pairs are either semi-conserved or conserved in all tRNAs suggesting that all tRNAs have similar frameworks.

Because of tRNA's high stability it easily renatures. The high degree stability is essential for the multi-functionality of tRNA. 71 out of 76 bases are stacked indicating base-stacking is primarily a stabilizing factor. Further more, nine tertiary base-pairs specifies the overall structural framework of tRNA. These base-pairs are either semi-conserved or conserved in all tRNAs suggesting that all tRNAs have similar frameworks. Another stabilizing factor is the ability of the 2' hydroxyl group to extensively hydrogen bond.

FUNCTIONAL IMPLICATIONS OF THE THREE-DIMENSIONAL STRUCTURES: Flexibility of the molecule. Refinement of thermal parameters of rigid groups such as base, ribose and phosphate from x-ray diffraction data illustrates the overall thermal flexibility of these groups. Results indicate unusually high thermal vibration of the acceptor stem and the anticodon arm and suggest that they are more flexible than the rest of the molecule or that the arms partially unwind. Thus slight movement of the acceptor stem and the anticodon arm during transpeptidation and translocation within the ribosome is feasible.

FUNCTIONAL IMPLICATIONS OF THE THREE-DIMENSIONAL STRUCTURES: Modified nucleosides. Base modifications are not essential to conserve the tertiary structure of tRNA, therefore strongly indicating that these modified bases are responsible for site recognition of various protein that interacts with tRNA. A modified 3' end of the anticodon allows only the codon-anticodon triplet base-pairing

FUNCTIONAL IMPLICATIONS OF THE THREE-DIMENSIONAL STRUCTURES: T-Y-C corner. The sequence G-T-Y-C-G in the T loops <Click button to view T stem (Yellow)> is found in all tRNAs participating in ribosome-meditated peptide elongation. Experimental evidence (Erdmann, 1976) indicates that the sequence C-G-A-A-C recognize the T loop sequence at the A-site of the ribosome. However the crystal structure shows that the T loop and the D loop is internally hydrogen bonded and cannot be available for rRNA recognition unless conformation changes occur. The same crystal structure shows that tRNA may undergo conformational changes when induced by 5S rRNA binding. A twist and a bend of 30° on base-pair G19-C56 suggest that these conformational changes are feasible.