Part:BBa_K1223014

tRNA-Pyl (pylT) gene from Methanosarcina barkeri str. Fusaro

this is the DNA coding sequence for the tRNA of pyrrolysine from the Archaea Methanosarcina barkeri str. Fusaro.

this tRNA is a part of the machinery that is used to incorporate pyrolysine into proteins in E.coli.

the tRNA synthetase that charges the tRNA with pyrrolysine can be found in biobrick BBa_k1223013.

the tRNA has the anticodon CUA - which means that the sense codon needed to incorporate pyrrolysine is TAG - the amber stop codon.

Cloverleaf representation of M. barkeri Fusaro tRNA^Pyl [9]

tRNAcua^pyl (pylT) gene from Methanosarcina barkeri:

This biobrick is the DNA coding sequence for the tRNAcua pyl of Pyrolysine from the Archaea Methanosarcina barkeri str fusaro. This tRNA is a part of the machinery that is used to incorporate pyrolysine (and other substrates) into proteins in bacteria. The PylRS that charges the tRNA with pyrrolysine can be found in biobrick BBa_k1223013. The tRNA naturally contains the anticodon CUA (Fig.4) – Thus it recognizes TAG as its sense codon when incorporating the UAA. With the introduction of this tRNAcua pyl from archaea into bacteria it can compete with the bacterial release factors in the recognition of the UAG codon of the mRNA. This competition can manipulate the bacterial translational machinery (stop codon suppression) and thus render the TAG non-sense codon (Amber stop codon) to a Sense codon for UAA incorporation.

UAA incorporation efficiency: For efficient stop codon suppression the expression of tRNA¬cua¬pyl levels should be high. For this reason the pylT gene must be cloned (along with the PylRS) into a high copy number expression plasmid. In addition, multiple copies (6-9) of the gene encoding for the amber suppressor (tRNAcua pyl) can be added to the same backbone.[6]

Design Notes

this is a part of the pyrrolysine incorporation machinery.

this tRNA is charged by pyrrolysyl-tRNA synthetase (pylS gene) to incorporate the amino acid pyro lysine. the tRNA has CUA anticodon that recognizes the TAG (UAG) codon on the mRNA during the translation process.

pylS CDS can be found in part BBa_K1223013.

Applications of BBa_K1223014

This part was used by us to incorporate the unnatural amino acid propargyl-L-lysine into various proteins. in the fluorescent gel we show incorporation of the unnatural amino acid into copper oxidase of E.coli in various loactions

H117,N262,D411,M412 - position and amino acid that was replaced with the UAA. CueO - native protein without UAA incorporated. L - ladder.

we used a reactive fluorescent dye to identify the incorporated UAA in the gel.

Potential applications of Genetic code expansion and UAA:

By the use of site specific incorporation of many different Unnatural amino acids into proteins numerous possibilities open up for synthetic biology and many other fields. Among those possibilities are:

1. Probes of Protein Structure and Function: Many biophysical and mechanistic studies require significant quantities of proteins with a probe incorporated at a unique site in a protein. UAA mutagenesis methodology is well suited to many such problems.[8]

2. Therapeutic proteins UAA mutagenesis is beginning to find many applications in the generation of therapeutic proteins, where the production of large quantities of homogenously modified protein is desired.[8]

3. Protein Evolution with an Expanded Genetic Code It is quite possible that the ability to encode additional amino acids with novel properties would be evolutionarily advantageous, especially since nature’s choice of 20 could have been arbitrarily fixed at the point of transition between communal and Darwinian evolution paradigms and subsequently sustained by the code’s inertia. Furthermore, in the limited scope of laboratory-directed evolution, which concerns only one or few specific functions over a short time rather than general organismal fitness over thousands or millions of years, one can easily envision a selective advantage conferred by additional amino acids. Because the templated assembly of polypeptides from mRNA on the ribosome establishes a direct link between genes (information) and proteins (phenotype), UAA mutagenesis methodology can easily be adapted to the evolution of proteins with novel or enhanced function.[8]

Source

derived from the genomic sequence of Methanosarcina barkeri. gene name - pylT

clasification: Archaea : Euryarchaeota : Methanomicrobia : Methanosarcinales : Methanosarcinaceae : Methanosarcina

References

[1] N. Budisa, “Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire.,” Angew. Chem. Int. Ed. Engl., vol. 43, no. 47, pp. 6426–63, Dec. 2004.

[2] L. Wang, J. Xie, and P. G. Schultz, “Expanding the genetic code.,” Annu. Rev. Biophys. Biomol. Struct., vol. 35, pp. 225–49, Jan. 2006.

[3] O. Nureki, Y. Nakahara, K. Nozawa, H. Hojo, and H. Katayama, “Pyrrolysine Analogs as Substrates for Bacterial Pyrrolysyl-tRNA Synthetase in Vitro and in Vivo,” Biosci. Biotechnol. Biochem., vol. 76, no. 1, pp. 205–208, 2012.

[4] J. M. Kavran, S. Gundllapalli, P. O. Donoghue, M. Englert, D. So, and T. A. Steitz, “Structure of pyrrolysyl-tRNA synthetase , an archaeal,” 2007.

[5] A. Théobald-Dietrich, M. Frugier, R. Giegé, and J. Rudinger-Thirion, “Atypical archaeal tRNA pyrrolysine transcript behaves towards EF-Tu as a typical elongator tRNA.,” Nucleic Acids Res., vol. 32, no. 3, pp. 1091–6, Jan. 2004.

[6] Y. Ryu and P. G. Schultz, “Efficient incorporation of unnatural amino acids into proteins in Escherichia coli,” vol. 3, no. 4, pp. 263–266, 2006.

[7] D. P. Nguyen, H. Lusic, H. Neumann, P. B. Kapadnis, A. Deiters, and J. W. Chin, “Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA Synthetase/tRNA(CUA) pair and click chemistry.,” J. Am. Chem. Soc., vol. 131, no. 25, pp. 8720–1, Jul. 2009.

[8] C. C. Liu and P. G. Schultz, “Adding new chemistries to the genetic code.,” Annu. Rev. Biochem., vol. 79, pp. 413–44, Jan. 2010.

[9] Sarath Gundllapalli, Alexandre Ambrogelly, Takuya Umehara, Darrick Li, Carla Polycarpo, Dieter Söll, Misacylation of pyrrolysine tRNA in vitro and in vivo, FEBS Letters, Volume 582, Issues 23–24, 15 October 2008, Pages 3353-3358, ISSN 0014-5793, http://dx.doi.org/10.1016/j.febslet.2008.08.027.

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]