Transfer RNA and its Interactions with Seryl-tRNA
Michael W. Hapiak, '99
Table of Contents:
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
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 , 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
The T-arm consists of a T-stem (five
base pairs; 49-53, 61-65) and T-loop (55-60).
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
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
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.
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.
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
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
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,
ii) there are no unpaired bases at the base of the variable
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. et.al. (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. et.al. (1994) The 2.9 Angstrom Crystal Structure
of T. thermophilus Seryl-tRNA Synthetase Complexed with tRNA
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
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.
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