Page 183 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

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Encyclopedia of Physical Science and Technology EN013D-617 July 27, 2001 11:42

Protein Synthesis 223

specifically and disregards the other 19 amino acids; it selection of the cognate tRNA by an AARS is based on
also recognizes tRNAs containing the anticodons GUN a limited number of sequence or structural elements, and
(where N is any nucleotide) and only attaches valine to in some cases does not involve the anticodon triplet of the
these molecules. genetic code.

A. Amino Acid Attachment
C. Amino Acid Selection
Covalent attachment of an amino acid to its cognate tRNA
Because of their small size and limited chemical diversity,
occurs in a two-step mechanism catalyzed by an AARS
selection of a cognate amino acid from the cellular pool
(E). In the first step, energy is consumed in the form of
presents a greater challenge to the AARSs than does se-
ATP to convert the enzyme-bound amino acid to its high-
lection of a specific tRNA. Differentiation of amino acids
energy aminoacyl adenylate (AA–AMP). In the second is primarily achieved through the formation of a binding

step the activated amino acid is transferred to the 3 end of
pocket specific for the cognate amino acid within the ac-
the cognate tRNA, where an ester bond is formed be-
tive site of the enzyme. In this way specific hydrogen bond
tween the carboxyl group of the amino acid and either
contacts can be made to a polar amino acid, or a positively

the 2 - or 3 -hydroxyl on the terminal adenosine’s ribose
charged amino acid can fit into a negatively charged cleft
sugar to yield AA–tRNA. While for most AARSs the first
within the AARS. Amino acids with hydrophobic side
step is tRNA-independent, glutaminyl–, glutamyl–, and
chains have to be discriminated on the basis of shape.
arginyl–tRNA synthetases require cognate tRNA binding
In some cases binding of cognate tRNA influences
for aminoacyl adenylate formation.
amino acid selection. Glutaminyl–tRNA synthetase
(GlnRS) is one of three AARSs that require the presence
E + AA + ATP E(AA–AMP) + PP i
of cognate tRNA for aminoacyl adenylate formation. Bio-
E(AA–AMP) + tRNA AA–tRNA + E + AMP. chemical and genetic studies demonstrated that conserved
residues of GlnRS that contact the 3 -terminus of tRNA Gln

are required to properly form the glutamine binding site.
B. Selection of tRNA
The presence of noncognate tRNAs decreased the
Because of the critical need for accurate tRNA amino- enzyme’saffinity for glutamine because only tRNA Gln
acylation in protein synthesis, AARSs must be highly dis- was properly oriented to allow formation of the glutamine
criminating in substrate selection. Structural studies re- binding pocket.
vealed a large area of contact between AARS and tRNA,
providing numerous opportunities for sequence-specific
D. Editing by AARSs
recognition, including contacts with the many modified
nucleotides of tRNAs. The majority of identity determi- Binding interactions alone can not completely discrimi-
nants lie in the anticodon and the acceptor stem (near nate against all noncognate amino acids. Some enzymes
the site of amino acid attachment) of the tRNA molecule. misactivate noncognate amino acids at a significant fre-
Although the genetic code links amino acid identity to quency and require an additional accuracy mechanism.
anticodon sequence, an “operational RNA code” exists The most well-characterized example is isoleucyl–tRNA
withintheacceptorstemsoftRNAs.ThisoperationalRNA synthetase (IleRS), responsible for activating isoleucine
Ile
code is considered by some to be a “second genetic code.” and attaching it totRNA at the exclusion of valine, which
It relates sequence and structure in the acceptor stem to is smaller than isoleucine by a single methylene group.
aminoacylation with specific amino acids. IleRS is more successful in discriminating against larger
Because determinants for aminoacylation are found or bulkier amino acids, but the smaller valine simply fits
in the acceptor stem, RNA minihelices are substrates more loosely in the enzyme active site.
for aminoacylation by at least 10 different aminoacyl– Despite misactivating valine as frequently as once per
tRNA synthetases. A striking example is alanyl–tRNA 180 correct (isoleucine) activations, the enzyme is able
synthetase, which makes no contact with the tRNA Ala an- to maintain the required fidelity of tRNA aminoacyla-
ticodon. Instead, the enzyme recognizes a unique G3:U70 tion through an active editing function, so that valine is
base pair in the acceptor stem of the tRNA. Substitutions not misincorporated into proteins to a significant extent.
at this location eliminate aminoacylation of the tRNA with This editing is twofold, corresponding to the two steps of
alanine, while introduction of this base pair into noncog- the aminoacylation reaction: pretransfer editing by IleRS
nate tRNA molecules target them for alanylation. An RNA selectively hydrolyzes the noncognate valyl–adenylate,
“minihelix” (Fig. 2) is also efficiently aminoacylated with while post-transfer editing hydrolyzes any valyl–tRNA Ile
alanine provided it contains the critical G3:U70 pair. Thus, that is formed.