Jihwan Jeon/scientific essay
Expanding genetic code for unstable non-canonical amino acids by using stop(amber) codon suppression technique
20141541 Jihwan Jeon
Introduction
The genetic code of all organisms encodes just 20 common amino acids. However, these 20 amino acids, canonical amino acids, can be modified by post-translation modification(PTM) process. Through the PTM tons of various amino acids are formed and they have their own important chemical biological functions in organism. Among those PTM, some of PTM’s function was still not discovered well. One example of that is N-phosphorylation. N-phosphorylation means phosphorylation to N group in side chain of His, Lys or Arg. Those N-phosphorylation was still quite not explored because of its low stability in N-phosphate bond.
DNA sequences are transcripted to mRNA and then translated to amino acids. During the translation, each three mRNA sequence is assigned to specific amino acid. In 2001 science, one interesting paper was published; Expanding genetic code of escherichia coli. In this paper, they generated unique tRNA/aminoacyl-tRNA synthetase pair to express non-canical amino acid at the site corresponed to amber codon in mRNA. In this way, they conducted in vivo incorporation O-methyl-tyrosine into protein in E-coli.
Then why don't we improve this technique to express some unstable non-canonical amino acids?
Body
Let's focus on N-phosphorylated amino acids as a subject for expanding genetic code. There were some previous researches about pHis and it is known that pHis conduct stress regulation system, so called two-component system, in E-coli. But still we don't know much about function of pHis in eukaryotic cell. Compared to other phosphorylated amino acids such as pTyr, pSer and pThr, N-phosphorylated amino acid were less understood because of acid-labile property.
N-phosphorylated amino acid are fragile because it undergoes hydrolysis in acidic or even neutral pH. Therefore most of in vitro phosphorylation to get pHis, pAsp and pArg in concucted at basic condition using potassium phosphoramidate.
Amber codon suppression is a technique to incorporate non-canonical amino aicds in protein by developing novel tRNA and aminoacyl-tRNA synthetase pair. Then if we can develop tRNA which carries N-phosphorylated amino acid in basic condition and develop matched aminoacyl tRNA synthetase, manual in vivo incorporation of N-phosphorylated amino acid in protein would be possible.
Colclusion
Chemical probe to catch N-phosphorylated protein have been developing and it is attractive way to figure out biological function of N-phosphorylation. I conducted reaearch about finding chemical probe of pLys and pAsp(althought it is not N-phosphorylation, it is also not stable) using antibody. This chemical approach to unstable phosphorylation is quite attractive but not easy. So I proposed another way to investigate the function of unstable non-canonical amino acids by developing tRNA and aminoacyl tRNA synthetase. If we can manage N-phosphorylated protein in organism, then it would be much easier, even after developing chemical probe for those amino acids, to conduct biological assay.
References
Kim Wals and HuibOvaa, Front. Chem., 01 April 2014, Unnatural amino acid incorporation in E. coli
P. G. Besant, P. V. Attwood, M. J. Piggott. Current Protein & Peptide Science, Volume 10 , Issue 6 , 2009, Focus on Phosphoarginine and Phospholysine
Lei Wang, Ansgar Brock, Brad Herberich, Peter G. Schultz, Science, Volume 292, 20 April 2001, Expanding the Genetic Code of Escherichia coli
