Transfer RNA and its Interactions with Seryl-tRNA Synthetase

Michael W. Hapiak, '99

Table of Contents:

I: Introduction

One of the most fundamental processes of molecular biology, crucial to the propagation of life, is that of RNA translation into protein. The Central Dogma outlines the mechanistic pathway through which genomic DNA is transcribed into mRNA, which then migrates out of the nucleus to designated sites within the cell where it can be translated into protein. It is this key principle which forms the theoretical basis for the complexity of all life processes.

 The mechanism of protein synthesis via translation is both specific and efficient. There exist numerous aminoacyl-tRNA synthetases, enzymes which activate tRNA molecules by facilitating the binding of a specific amino acid through an esterification reaction. Each one is engineered to specifically recognize the codon site of mRNA which encodes for the insertion of its specific amino acid. Because the genetic code is degenerate, there exist many different synthetases which can specify for one type of amino acid. Yet due to site-specificity of anticodon recognition, translation is very precise.

 Here we will examine the structures of transfer RNA and seryl-tRNA synthetase, as well as the interactive complex they form with eachother during translation. Through an investigation of structural motifs and binding specificities we will examine the mechanism of serine incorporation into a growing polypeptide chain.

II: Transfer RNA

Transfer RNA is a fairly small molecule, consisting of 75-90 nucleotides, which folds over itself (via hydrogen bonding between base pairs at four different sites), forming a three-dimensional L-shaped structure. The double stranded sites are referred to as stems , and the non-bonded sites as loops <>. The tRNA molecule is made up of four general regions: the acceptor arm , the T-arm , the anticodon arm , and the D-arm. Another less significant region is the variable loop <>.

In the tRNA, the acceptor stem is made up of seven base pairs (1-7, 66-76), and at the 3' end of the acceptor stem is the amino acid attachment site (74-76) <>. This is where the synthetase molecule activates the tRNA by attaching serine. The acceptor stem is believed to carry the primary identity elements for recognition.

The T-arm consists of a T-stem (five base pairs; 49-53, 61-65) and T-loop (55-60). The T-loop <> is so named because it contains thymine , a nucleotide that is found in DNA but not in any other RNA species besides tRNA . The anticodon arm <> is comprised of the anticodon stem (five base pairs; 27-31, 39-43) and its corresponding loop region (32-38). The anticodon region  is where the anticodon triplet base-pairs with the codon triplet of the messenger RNA.

The D-arm is made up of the D-stem (four base pairs; 10-13, 22-25) and the D-loop (14-21). This region is so named because it contains the modified base dihydrouracil. The D-stem is also believed to contains some identity elements for recognition. A major feature of the D-loop is a "zig-zag" conformation of the backbone, specifically caused by a bulging in of the G20 residue <>. The variable loop (44-48) protrudes at approximately a 450 angle to the plane of the L-shaped tRNA <>.

III: Seryl-tRNA Synthetase

Class 2 tRNA synthetases have two domains: an N-terminal nucleotide binding fold which is the active site for interaction with the acceptor stem of tRNA, and a C-terminal <> domain which associates with the anticodon arm of the tRNA molecule. The N-terminal domain is comprised of an exceptionally long, solvent exposed anitparallel coiled-coil helical arm <>. Residues within this structure that interact with the variable stem of tRNA involve Lys542, Gln548, Thr549, and Asn552 <>. Due to these features, the synthetase has the ability to simultaneously bind two tRNA molecules at a time.

Seryl-tRNA synthetase has at least five distinct species which collectively recognize six different codons for the amino acid. This suggests that anticodon recognition is not the primary recognition determinant, and that serine specificity is guaranteed by two structural characteristics of the synthetase molecule: i) two distinct hydrogen-bond interactions with the amino acid sidechain hydroxyl group, and ii)the limited size of the synthetase's binding pocket, which cannot accomodate any other sidechains larger than the hydroxyl. Secondary specificity is achieved through descrimination during the aminoacylation transition state of the complex. The complexes are short-lived, and this facilitates rapid turnover and greater specificity action. The catalytic domain is made up of a seven-stranded antiparallel beta sheet <> with two connecting helices.

IV: tRNA-Synthetase Interactions

Several tertiary interactions are observed within tRNA involving the hydroxyl groups of the sugar riboses (which is particularily interesting considering that these -OH groups are missing in the DNA sugar molecules), and these hydroxyl groups are essential for stabilizing the coiling action of tRNA. Another characteristic structure is the noticed "wobble" <>, which is caused by the mispairing of a guanine and uracil, resulting in a slight distortion of the molecule's structure. Also, a mechanistic action observed during protein synthesis is the movement of the D-loop and T-loop away from eachother as the molecule shifts from the A site of the ribosome complex to the P site during the transcription process .

 The long variable arm exhibits an orientation determined by the interactions of the antiparallel coiled-coil motif of the synthetase with the tRNA core <>. These interactions include:

i) tRNA binding of two subunits across the dimer,

 ii) the stabilization of the coiled-coil of the synthetase (upon binding), curving between the T-loop and variable loop,

 iii) multiple backbone contacts but few base-specific interactions,

 iv) contacts extend out to the sixth base pair, and

v) bases G20a and G20b are inserted into the D-loop <>.

G20 is stacked against the first base pair of the long variable arm, determining its orientation. Two features of major importance for synthetase recognition of any given variable loop are:

 i) the number of unpaired bases at the beginning of the variable arm stem correlate with the number of inserted nucleotides in the D-loop, and

 ii) there are no unpaired bases at the base of the variable arm stem.

ATP Binding

Characteristic interactions of ATP binding to motif 2 of the synthetase molecule include a stacking interaction with the Phe275 residue, Arg256 association with an alpha-phosphate, and Glu345 (from another beta strand) interaction with the ribose 3' hydroxyl <>. These mechanisms allow for the activation and propagation of the translation mechanism of protein synthesis.


Belrhali, H. (1994) Crystal Structures at 2.5 Angstrom Resolution of Seryl-tRNA Synthetase Complexed with Two Analogs of Seryl Adenylate. Science 263: 1432-36.

 Biou, V. (1994) The 2.9 Angstrom Crystal Structure of T. thermophilus Seryl-tRNA Synthetase Complexed with tRNA Ser. Science 263: 1404-10.

 Eriani, G. (1990) Partition of tRNA Synthetases into Two Classes Based on Mutually Exclusive Sets of Sequence Motifs. Nature 347:203-6.

 Gale, A. and Schimmel, P. (1995) Isolated RNA Binding Domain of a Class I tRNA Synthetase. Biochemistry 34:8896-8903.

Rich, A. and Kim, S. The Three Dimensional Structure of Transfer RNA.Science America.238: 52-62.

 Schimmel, P. (1989) Parameters for the Molecular Recognition of Transfer RNAs. Biochemistry 28: 2747-58. 

Back to Molecules Index