By Patrick Ginther

A standing goal of synthetic biology, and especially of the Center for Genetically Encoded Materials, is to engineer an organism with specialized ribosomes that can encode unnatural polymers with new and interesting functions. One challenge in this effort is that, because ribosomes make all the proteins in a cell, changing ribosomes to give them new functions is usually toxic. To get around the toxicity that usually comes from engineering ribosomes, they can be made orthogonal to endogenous ribosomes, allowing them to be mutated without affecting the cell.

Ribosomes can be made orthogonal by changing the anti-Shine-Dalgarno sequence so that it only interacts with a corresponding orthogonal Shine-Dalgarno sequence.[1] However, this change only makes the small subunit orthogonal, as large subunits still exchange freely through the cell. To address this issue, two labs independently developed a method of tethering the subunits of the ribosome together, effectively isolating the orthogonal ASD to a single ribosome.[2,3] Initially, this method was thought to effectively produce an orthogonal ribosome. However, the Chin lab has recently identified trans interactions between endogenous and tethered ribosomes in which the tethered ribosomes open up to allow cross-assembly with endogenous ribosomal subunits.[4] These cross-assembly interactions mean that engineered tethered ribosomes could still prove toxic to cells.

In this recently published Nature paper, the Chin lab was able to eliminate these unfavorable cross-assembly interactions by altering the length of the tether which links the two subunits together. First, they generated a small library of insertions or deletions on either side of the tether. Next, they examined how much cross-assembly occurred via an in vitro GFP translation assay.[4] From this, the lab identified variant d2d8, which showed the highest activity with a minimal amount of cross-assembly.

To illustrate the utility of a truly orthogonal ribosome, the Chin lab then engineered a version of the tethered ribosome d2d8 that can read through polyproline sequences—something that endogenous ribosomes can’t do without help from elongation factor P (EF-P). After screening for mutants with changes in both the peptidyltransferase center and the ribosomal exit tunnel, they found a mutant—d2d8 (5)—that was able to effectively read through 7 prolines in a row without EF-P.[4] This example illustrates the exciting potential of an orthogonal tethered ribosome to engineer new, interesting functionality.

1. Rackham, O., Chin, J., Nature Chemical Biology, 2005, 1, 159–166.
2. Orelle, C. et al. Nature, 2015, 524, 119-12.4
3. Fried, S. et al. Angew. Chem. Int. Ed,. 2015, 54, 12791-12794
4. Schmied, W. et al. Nature, 2018, ASAP.