Method for tracking orthogonal ribosomes in vivo using MS2coat protein
Abstract: Ribosomes are large macromolecules responsible for protein synthesisand they consist of both RNA and proteins. Each ribosome is made of one large and one small subunit. Even though the ribosome is one of the most studied machineries in the cell there is a gap in our understanding of how this macromolecule functions in vivo. In this project we aimed to develop a method for tracking a specific subset of ribosomes using super-resolution fluorescence microscopy. This was achieved by using the MS2 coat protein (MS2CP) fused to a fluorescent marker and by modifying ribosomes to have the RNA loop to which theMS2CP binds with high affinity. We were able to obtain the most promising results when the MS2CP was fused to a Halotag with the dye JF549 attached to it. The JF549 has good cell permeability which allows simple and efficient labelling of ribosomes. To be able to observe translation of specific mRNAs we used this labelling strategy to track orthogonal ribosomes which do not recognise mRNA normally produced in cells but can translate mRNAs with a modified 5’-UTR.Orthogonal ribosomes were tested with several different 5´-UTRs. With the construct for which we obtained the highest expression level we observed that up to 43% of the labelled orthogonal ribosomes were engaged in translation of the specific mRNA. The system will make it possible to determine how the sequence of a particular mRNA will affect its in vivo translation.
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