Beruflich Dokumente
Kultur Dokumente
Vipin Shankar
The Central Dogma
Translation : overview
PPi
Charging…
As a result of adenylylation, the amino acid
is attached to adenylic acid via a high-
energy ester bond in which the carbonyl
group of the amino acid is joined to the
phosphoryl group of AMP.
The adenylylated amino acid remain tightly
bound to the synthetase.
Charging…
Step 2: adenylylated amino acid is
transferred from the enzyme to the 3’ end of
the tRNA via the 2’- or 3’ – hydroxyl and
the concomitant release of AMP.
There are 2 classes of tRNA synthetases.
Class I enzymes attach amino acids to 2’ OH of
tRNA and are generally monomeric.
Class II enzymes attach amino acids to 3’ OH
of tRNA and are typically dimeric or trimeric.
General Structure of amino acyl tRNA
Charging…
Each of the 20 amino acids is attached to the
appropriate tRNA by a single, dedicated tRNA
synthetase.
Because, most amino acids are specified by more
than one codon, it is not uncommon for one
synthetase to recognize and charge more than one
tRNA: Isoaccepting tRNAs.
Nevertheless, the same tRNA synthetase is
responsible for charging all tRNAs of a particular
amino acid.
Specificity of charging
tRNA synthetases:
Must recognize the correct set of tRNAs for a
particular amino acid, (specificity of tRNA
recognition) and
must charge all these isoaccepting tRNAs with
the same amino acid (Accuracy of amino acyl
tRNA formation).
Specificity of tRNA recognition
tRNA synthetases recognize some specific
regions of the tRNA which help them to
identify the correct cognate tRNA –
specificity determinants.
Specificity determinants are clustered at 2
distinct sites on the tRNA-
the acceptor stem - discriminator.
the anti-codon loop – anti-codon.
Specificity of tRNA recognition…
The anti-codon dictates the amino acid, that the
tRNA is responsible for incorporating.
However, because each amino acid is usually
specified by more than one codon, recognition of
anti-codon cannot be used in all cases.
So the discriminator plays a greater role in tRNA
recognition.
The set of tRNA determinants that enable
synthetases to discriminate among tRNAs is
referred to as ‘the second genetic code’.
Accuracy of amino acyl tRNA formation
The challenge to recognize the correct
amino acid is even more daunting.
The relatively small sizes and the similarity
makes the task difficult.
Despite this challenge, the frequency of
mischarging is very low: typically less than
1 in 1000 tRNAs is charged with incorrect
amino acid.
Accuracy of amino acyl tRNA formation...
Different synthetases use different
mechanisms to distinguish between the
amino acids.
Eg 1.
Tyrosyl tRNA synthetase: the oppertunity of
forming a strong and energitically favourable
hydrogen bond with the hydroxyl moiety of
tyrosine helps the synthetase to identify
tyrosine.
Accuracy of amino acyl tRNA formation...
Eg 2.
Isoleucine and valine differ only by a single methyl
group.
Valyl tRNA synthetase can sterically exclude
isoleucine from its catalytic pocket as isoleucine is
larger than valine.
Eg 3.
But valine can easily slip into the isolucyl tRNA
synthetase catalytic pocket.
The interaction of the methyl group on isoleucine gives
an extra -2 to -3 Kcal/mol of free energy. This
relatively small energy difference makes the reaction
100 times more likely.
Accuracy of amino acyl tRNA formation...
One common mechanism to increase fidility of an
amino acyl tRNA synthetase is to proofread the
products of the charging reaction.
This is performed by an editing pocket present in
addition to the catalytic product.
All wrongly charged amino acyl tRNAs enter the
editing pocket, while the correctly charged amino
acyl tRNA is sterically excluded (molecular
sieve).
Within the editing pocket the amino acyl tRNA is
hydrolyzed.
Accuracy of amino acyl tRNA formation...