Part:BBa_K3009032
Basic Part: ms2 loop BBa_K3009032 Group: Freiburg Author: Alisa Summary: MS2 stem loop structures can be fused to the untranslated 3`end of an mRNA sequence. The MS2 Capsid Protein possesses a strong binding affinity towards the loops. A protein-mRNA complex is built within the cytosol, which also brings fusion proteins of MCP close to the mRNA. Documentation:
Usage
The MS2 system is a tool to connect mRNA sequences and proteins with each other. The mRNA is tagged with the phage-derived stem-loops, which build an mRNA-Protein complex with MCP, which binds to the loops.[1] Fusion proteins of MCP, like fluorescent reporter proteins and orthogonal tRNA Synthetases can this way be brought into closer proximity of the tRNA. [2]
Biology
stem-loops are part of the regulatory system of the MS2 phage which is active in early infection stages, and block replicase activity by binding the MS2 coat protein. [3]
Characterization
Backbone: pULTRA
Promoter: lacI, proK E.coli strain: C321.deltaA
We tagged various sfGFP constructs containing amber stop codons with three repeats of the ms2 loop structures to bring an orthogonal tyrosyl-tRNA-Synthetase in close proximity of the sf GFP mRNA. A noncanonical amino acid can therefore be incorporated instead of the suppressed stop codon.
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.
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