Extended Data Figure 1. Partitioning of free large subunits between wild-type and orthogonal small subunits, and in vivo activity of O-stapled ribosomes.

a Gain-of-function mutations in free large subunits may confer gain-of-function phenotypes through statistical partitioning of free large subunits between wild-type (WT) and orthogonal small subunits. A mutant large subunit (LSU) can partition between WT and orthogonal (green) small subunits (SSUs) in cells that contain both WT large subunits (not shown) and mutant large subunits. Hence the mutant phenotype will be observed in the translation of both WT and orthogonal messages (see Extended Data Fig. 4d for an example). b In vivo activity of linker length variants of O-stapled ribosomes (same data set as Fig. 2b). GFP expression was analysed in E. coli cells containing the indicated O-ribosome, the Methanosarcina mazei PylRS synthetase/tRNACUA pair, and the O-sfGFP150TAG reporter, in the presence of 1 mM BocK (N-epsilon (tert-butoxylcarbonyl)-l-lysine). GFP fluorescence is shown as a percentage of that produced from an orthogonal ribosome with independent, non-linked subunits. O-d2d8 is highlighted in blue, and O-ribosomes with previously described subunit linkers are in dark grey. Statistics are detailed in the Methods.