Part:BBa_K3009031

Usage

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. [1] 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. [2]

Biology

The MS2 Capsid Protein performs regulatory functions in the early stages of an MS2 bacteriophage infection of ... 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 Backbone: pULTRA Promoter: lacI, proK E.coli strain: C321.deltaA

We utilized the MCP-ms2 loop binding to bring an orthogonal tyrosyl-tRNA Synthetase into close proximity of a superfolder GFP mRNA containing an amber stop codon mutation at position 66. The otherwise low-yielding reaction should be greatly improved by the MS2 system.

Fig. 1:Comparison of fluorescence intensity in C321.deltaA E.coli cells expressing ms2-tagged sfGFPY66* with and without co-expression of MCP-tagged tyrosyl-tRNA Synthetase.

References

[1] Haimovich, et al. (2016): Use of the MS2 aptamer and coat protein for RNA localization in yeast: A response to "MS2 coat proteins bound to yeast mRNAs block 5' to 3' degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system". In: RNA (New York, N.Y.) 22 (5), S. 660–666 [2] Reinkemeier et. al 2019): Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. In: Science (New York, N.Y.) 363 (6434).

[3] Peabody, D. S. (1993): The RNA binding site of bacteriophage MS2 coat protein. In: The EMBO Journal 12 (2), S. 595–600.