Fluorescent proteins misreport ribosome abundance under translation inhibition

Fluorescent proteins, commonly employed as quantitative indicators of protein abundance in living cells, may not always accurately reflect ribosome abundance during translation inhibition. This phenomenon was demonstrated in a study involving Escherichia coli and Bacillus subtilis. The researchers found that while fluorescence intensity generally tracks ribosome abundance under nutrient-limited growth, this correlation breaks down during chloramphenicol treatment.

The discrepancy was observed with both transcriptional and translational reporters, as well as with both ribosomal protein and ribosomal RNA promoters. To differentiate between changes in fluorescence output per reporter and changes in reporter abundance itself, the team created a dual fluorescent protein-LacZ reporter. Under translation inhibition, the LacZ-derived reporter abundance increased, while fluorescence intensity remained approximately constant.

This indicates that fluorescence output per unit reporter abundance decreases during translation inhibition.

The study also found that this effect is not limited to a specific fluorescent protein or translation inhibitor. In a related study, Bakshi and colleagues showed that the phenomenon extends to other fluorescent proteins and various translation inhibitors, suggesting that it is not a protein- or drug-specific issue. The researchers conclude that fluorescent-protein calibration can be condition-dependent and cannot be assumed to transfer across physiological perturbations.