Michael W. Hapiak, '99
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.
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 <>.
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
with two connecting helices.
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
Biou, V. et.al. (1994) The 2.9 Angstrom Crystal Structure of T. thermophilus Seryl-tRNA Synthetase Complexed with tRNA Ser. Science 263: 1404-10.
Eriani, G. et.al. (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.