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9. Beskrive hvordan tRNA aminoacyleres (charges) i 3'-enden med en aminosyre,
herunder hvordan følgende elementer indgår i processen: korrekt (cognate) tRNA,
aminosyre, aminoacyl-tRNA sythetase og ATP
Devlin, s.239-242
Stryer, s.817-822, fig. 29.7
In order to be incorporated in proteins, amino acids must first be activated by linkage to their appropriate tRNA carriers. This two-step process is catalysed by a family of aminoacyl-tRNA synthetases, called activating enzymes.
I will describe the catalysed process stepwise:
The amino acid reacts with ATP. The carboxyl group of the amino acid gets linked to the α-phosphate group of AMP – aminoacyl - AMP.
The aminoacyl-group of aminoacyl – AMP is then transferred to a particular tRNA, forming an aminoacyl – tRNA in which the a. a carboxyl group is linked to the OH-group of adenylate ribose.
Two molecules of ATP are consumed in the process; one in forming the ester linkage of aminoacyl t-RNA; the second one in driving the reaction forward.
The linkage of an amino acid to a tRNA is crucial for 2 reasons:
An attachment of
an amino acid to a particular tRNA establishes the genetic code. When an amino
acid has been linked to a tRNA, it will be incorporated into the growing
polypeptide chain at a position dictated by the anticodon of tRNA.
The formation of a peptide bond between free amino acids is not thermodynamically favourable. The amino acid must first be activated for protein synthesis to proceed. The activated intermediates in protein synthesis are amino acid esters in which the carboxyl group of an amino acid is linked to either the 2’- or 3’-OH group of the ribose of the adenylate residue of the acceptor stem CCA.
Aminoacyl t-RNA – an amino acid ester of tRNA. Only one amino acid is attached by an ester bond to an OH-group of the adenylate ribose.
Polypeptidyl t-RNA – polypeptide ester of tRNA. An entire polypeptide chain is attached to the ribose.
The correct selection of both tRNA and the amino acid by synthetase is central to fidelity in protein synthesis. It has been established that the aminoacyl-tRNA synthetases have separate structural domains that are involved in the catalytic process, tRNA and amino acid recognition.
1. Selection of the correct amino acid – some amino acids are easily recognized by their bulk, lack of bulk or by positive or negative charges on their side chains. Other amino acids, which differ only in an OH- or CH3 -group are more difficult to discriminate. If the amino acids differ in their chemical properties but are very similar structurally, the enzyme contains a jon in its binding site that can interact with fx. the OH-group but not with the methyl group. (threonine and valine)
If the two amino acids have similar chemical properties and almost same structure, an additional proof-reading step is required because the synthetase can not discriminate between the two.
The error rate of synthetase is ca. 1 in 10 4, 5 after the proofreading.
2. Selection of the correct tRNA – tRNA molecules are more complex then amino acids, so it should be easier to recognise them, even though they have the same basic cloverleaf structure. The right amino acid can be recognized by:
· their anticodon loop, when base pairs in the loop form hydrogen bonds with specific part of the enzyme
· base pairs in their acceptor stem (several nucleotides besides the CCA sequence)
· parts of the variable loop
· micro-helix, containing only 1/3 of the native tRNA can sometimes be enough
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