Part:BBa_K3009037
synthetase with MCP
Group: Freiburg
Author: Alisa
Summary: The tyrosyl-tRNA-synthetase from M.jannashii and cognate tRNA pair are acting orthogonally to the endogenous translational machinery of E.coli. By binding and loading non-canonical amino acids onto the tRNA, the tyrosyl-tRNA-synthetase enables their incorporation into proteins. The tRNA is cognate towards the amber stop codon, which is suppressed by the non-canonical amino acid. [1]
MS2 Capsid Protein(MCP), binds strongly to specific stem-loop structures derived from the MS2 phage genome. Proteins fused to MCP can be brought in close proximity to mRNA tagged with stem-loops.[3]
Documentation:
Usage
The tyrosyl-tRNA-synthetase was engineered to recognize and load para-cyano-phenylalanine onto its cognate tRNA, which likewise has been mutated to be complementarity towards the amber stop codon. [1] The synthetase exhibits an unusually high substrate promiscuity. Being able to incorporate over 20 distinct non-canonical amino acids, with several D-amino acids among them, onto the tRNA. [2] MCP, along with a fusion protein, can bind to an mRNA sequence tagged with specific stem-loops. This system is usually used for in vivo mRNA visualization by fusing MCP with a fluorescent reporter protein. [3] It can also be fused to an orthogonal aminoacyl tRNA synthetase used for the incorporation of noncanonical amino acids into proteins. The synthetase can be brought into close proximity of the stem-loop tagged mRNA of the protein of interest. [4]
Biology
In nature, the tyrosyl-tRNA-synthetase catalyzes the bond between the tyrosyl-tRNA and tyrosine in the archaea Methanocaldococcus jannaschii. The tRNA can then bind to the complementary codon of a translated mRNA sequence and enable the incorporation of a tyrosine into the protein.[2] The MS2 Capsid Protein performs regulatory functions in the early stages of an MS2 bacteriophage infection It forms a protein-RNA complex with a specific RNA stem-loop structure derived from the phage genome. The original function of the MS2 Capsid Protein is to stabilize RNA to prevent replicase synthesis and enable the encapsidation of the genome.[3]
Characterization
The bimolecular fusion of MCP and M. jannashii tyrosyl-tRNA Synthetase was used to incorporate D-Phenylalanine into the chromophore and a permissive site of GFP in response to an amber stop codon mutation.
References
[1]Chatterjee, et. Al, (2013): A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli. In: Biochemistry 52 (10), S. 1828–1837
[2]Ma, Hairong et. al (2015): Genetic incorporation of d -amino acids into green fluorescent protein based on polysubstrate specificity. In: RSC Adv. 5 (49), S. 39580–39586.
[3]Peabody, D. S. (1993): The RNA binding site of bacteriophage MS2 coat protein. In: The EMBO Journal 12 (2), S. 595–600
[4]Reinkemeier et. al (2019): Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. In: Science (New York, N.Y.) 363 (6434).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1107
Illegal EcoRI site found at 4055
Illegal XbaI site found at 3608 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1107
Illegal EcoRI site found at 4055
Illegal NheI site found at 1689
Illegal NheI site found at 3572
Illegal NheI site found at 3595
Illegal NheI site found at 4049 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1107
Illegal EcoRI site found at 4055
Illegal BamHI site found at 3988
Illegal XhoI site found at 4509 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1107
Illegal EcoRI site found at 4055
Illegal XbaI site found at 3608 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1107
Illegal EcoRI site found at 4055
Illegal XbaI site found at 3608
Illegal NgoMIV site found at 2622
Illegal NgoMIV site found at 5917
Illegal AgeI site found at 503
Illegal AgeI site found at 1775
Illegal AgeI site found at 2099
Illegal AgeI site found at 3120
Illegal AgeI site found at 3823 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 4981
Illegal BsaI site found at 5605
Illegal SapI site found at 3805
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 932
Illegal XbaI site found at 1041
Illegal PstI site found at 944
Illegal PstI site found at 1014
Illegal PstI site found at 1053 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 944
Illegal PstI site found at 1014
Illegal PstI site found at 1053 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 932
Illegal XbaI site found at 1041
Illegal PstI site found at 944
Illegal PstI site found at 1014
Illegal PstI site found at 1053 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 932
Illegal XbaI site found at 1041
Illegal PstI site found at 944
Illegal PstI site found at 1014
Illegal PstI site found at 1053 - 1000COMPATIBLE WITH RFC[1000]
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