Difference between revisions of "Part:BBa K1223013"

Latest revision as of 14:20, 2 November 2013

Pyrolysyl-tRNA synthetase CDS

this part is the coding sequence for the Pyrolysyl-tRNA synthetase enzyme from the archaea Methanosarcina barkeri str. fusaro. The enzyme 'load' the amino acid pyrolysine onto its dedicated tRNA for subsequent incorporation into proteins during the translation process. The tRNA synthetase is part of the unnatural amino acid (UAA) incorporation machinery along with the dedicated tRNA molecule (BBa_K1223014).

Design Notes

CDS of the pyrolysyl-tRNA synthetase (pylS) that charges the pyrolysine tRNA (pylT) with pyrrolysine.

Applications of BBa_K1223013

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.

Source

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

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

Genetic code expansion using stop codon (Amber) suppression in bacteria

Natural translation process is achieved via conservative mRNA-tRNA codon-anticodon specific base pairing; the meaning of each codon is interpreted mainly through stringent substrate specificity of Aminoacyl tRNA synthetases (AARS) in the aminoacylation reaction. This reaction is an interpretation level in the context of the flow of genetic information transmission.[1] In order to incorporate the #21 man made, synthetic Unnatural amino acid (UAA) one must first find a way to expand the genetic code to add a translational sense codon for that amino acid. We used a method called "Stop codon suppression"[2] developed by Prof. Peter Schultz and co-workers. This method uses orthogonal tRNAcua and Aminoacyl-tRNA synthetase from archaea. Orthogonal means that these components do not interact with the different host organism’s cellular pathways.

Orthoginality:

Since the PylRS originating from archea must be orthogonal in the host cell, our BioBricks could and should be used only in bacteria [2]. There is no orthogonality between archea and mammalian cells for example.

Use in our project:

The following parts were used by us to incorporate the unnatural amino acid proparagyl-L-lysine (as a model UAA) into our PASE 2 essential protein – TyrRS. When one of the following parts or the UAA itself are missing, one of the logical AND gate (Link to PASE2 project description) conditions are not met and essential protein translation is disabled. Pyrolysyl tRNA synthetase (PylRS) from Methanosarcina barkeri str. Fusaro (Archea): In well-studied model organisms such as E.coli , yeast and in mammalian cells, the natural AARS catalyse via the aminoacylation reaction the amino acid activation and accurate biosynthesis of aminoacyl-tRNA, the immediate precursors for encoded proteins. Note that each AARS should select its "own" (i.e cognate) amino acid (AA) which is subsequently covalently linked to the cognate tRNA isoacceptor "Fished" from the cellular pool. Immediately after their dissociation from AARS, the aminoacyl tRNA are shuttled or channeled to the ribosome where the anticodon is matched to the mRNA codon and the tRNA is deacylated , with the amino acid being added as the next residue of the a nascent protein chain.[1] In our project we used a class 2 AARS that is capable of charging Pyrolysine (Pyl – a natural, but rare, amino acid) and several unnatural Amino acids (Fig.1) onto the tRNAcuapyl.

PylRS substrates:

The PylRS can use different derivatives of Pyrolysine with high specificity and fidelity, The substrates of the PylRS are [3]:

As can be seen in Fig. 2 the aminoacylation of the tRNA by the PylRS is specific and selective to the latter substrates[3].

In Fig.2 tRNA were incubated with various lysine derivatives and PylRS, Next all samples were subject to acidic PAGE and stained with methylene blue. All samples that have 2 bends have been aminoacylated with the relevant lysine derivative. In our study proparagyl-lysine (figure 1, (8)) was used as a model amino acid.

PylRS Structure:

The crystal structure of the entire protein still eludes structural biologists but the crystal structure of its catalytic domain has been recently solved (Fig.3). The PylRS catalytic domain structure analysis reveals that it utilizes a deep hydrophobic pocket for recognition of the Pyl side chain (The pocket binds both tRNA and the amino acid and catalyzes the aminoacylation reaction between them) [4].

This Biobrick is the coding sequence for the Pyrolysyl tRNA synthetase enzyme from the archaea Methanosarcina barkeri str. fusaro. As mentioned earlier, the enzyme selectively 'loads' the amino acid, pyrolysine (and other synthetic substrates) onto its cognate tRNA for subsequent incorporation into proteins during the translation process in the ribosome. The tRNA synthetase is part of the unnatural amino acid (UAA) incorporation machinery along its cognate tRNA molecule (BBa_K1223014).

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]

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.

Usage and Biology

Sequence and Features