ARTIFICIAL SYNTHESIS OF A GENE FOR BACTERIAL TYROSINE tRNA

In 1975, Khorana and co-workers completed the synthesis of a gene for E.coli tyrosine
t RNA precursor. E.coli t RNA precursors are formed from the larger precursors. The tyrosine t RNA precursor has 126 nucleotides. They sunthyesized the complete sequence of DNA duplex coding for tyrosine –t RNA precursor of E.coli. though these segments are not the proper structural gene yet are the regions involved in its regulation.

Twenty six small oligonucleotide DNA segment giving rise to t RNA precursor was synthesized which were arranged into six double stranded fragments each containing single stranded ends. These six fragments were joined to give rise complete gene of 126 base pairs for tyrosine t RNA precursor of E. coli.

Khorana completely synthesized a biologically functional tyrosine t RNA suppressor gene of E.coli which was 207 base pairs long and contained (i) a 51 base pairs long DNA corresponding to promoter region, (ii) a 126 base pair long DNA corresponding to precursor region of t RNA, (iii) a 25 base pair long DNA including 16 base pairs contained restriction site for Eco RI. This complete synthetic gene was joined in phage lambda vector which in turn was allowed to transfect E.coli cells. After transfection phage containing synthetic gene successfully multiplied in E.coli.

Khorana made the phosphodiester approach for synthesizing the oligonucleotides of the biologically active t RNA. The demerits of this approach are: (i) the completion of reaction in long time, (ii) rapidly decrease in yield with the increase in chain length, and (iii) time taking procedure of purification.