Question

You will investigate how unnatural amino acid mutagenesis works. Take time to examine what changes needed to be made to the tRNA to allow for an unnatural amino acid to be used instead of the native substrate. Also focus on what codons are used to recognize the modified tRNA. How many different codons are there that can be used? What are they called?

Answer 1

Non sense suppression is the method to introduce unnatural amino acids to proteins. It involves charging suppressor tRNA with a synthetic amino acid. Suppressor tRNA arise naturally by mutation in the anticodon region of a wild type tRNA. Mutation causes suppressor tRNA to recognize stop codons. for example the wild-type tyrT gene encodes a tRNA that recognizes a 5' UAC 3' codon in the mRNA and inserts tyrosine into the growing polypeptide chain. A mutation in the gene changes the anticodon so that it recognizes the stop codon 5' UAG 3' in the mRNA and, instead of terminating, inserts a tryrosine at that position in the polypeptide chain.

This method of non sense suppression involves modification in mRNA and tRNA.

1 Modification of mRNA

Codon of a particular amino acid which has to be changed in the protein is replaced by non sense (stop codon). Translation of such modified mRNA will make a truncated protein. If the cells are supplied with a modified tRNA, which can recognize the stop codon and carries a modified/unnatural amino acid, translation will continue. Thus leading to incorporation of an unnatural amino acid.

2 Modification of tRNA

tRNA has to be modified to recognize stop codon and carry an unnatural amino acid on the 3^' end of its acceptor arm. Therefore, mutation in the anticodon is needed to recognize stop codon.

Desired tRNA is prepared by chemically coupling dCA dinucleotide with the unnatural amino acid. The dCA-amino acid conjugate is then joined to rest of the tRNA body by enzymatic ligation using T4 DNA ligase.

Stop codons like UAA (ochre) , UGA (opal), UAG (amber) can be used to recognize the modified tRNA.