Evolved orthogonal ribosome purification for in vitro characterization.
We developed orthogonal ribosome-mRNA pairs in which the orthogonal ribosome (O-ribosome) specifically translates the orthogonal mRNA and the orthogonal mRNA is not a substrate for cellular ribosomes. O-ribosomes have been used to create new cellular circuits to control gene expression in new ways, they have been used to provide new information about the ribosome, and they form a crucial part of foundational technologies for genetic code expansion and encoded and evolvable polymer synthesis in cells. The production of O-ribosomes in the cell makes it challenging to study the properties of O-ribosomes in vitro, because no method exists to purify functional O-ribosomes from cellular ribosomes and other cellular components. Here we present a method for the affinity purification of O-ribosomes, via tagging of the orthogonal 16S ribosomal RNA. We demonstrate that the purified O-ribosomes are pure by primer extension assays, and structurally homogenous by gel electrophoresis and sucrose gradients. We demonstrate the utility of this purification method by providing a preliminary in vitro characterization of Ribo-X, an O-ribosome previously evolved for enhanced unnatural amino acid incorporation in response to amber codons. Our data suggest that the basis of Ribo-X's in vivo activity is a decreased affinity for RF1.
Barrett
Oliver P T
OP
Medical Research Council Laboratory of Molecular Biology, Hills Roads, Cambridge, CB2 OQH, England, UK.
Chin
Jason W
JW
D002458:Cell Fractionation; D019020:Directed Molecular Evolution; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000378:metabolism; D014176:Protein Biosynthesis; D012336:RNA, Ribosomal, 16S / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*
D010454:Peptide Termination Factors; D012336:RNA, Ribosomal, 16S