Deciphering the Rules of Ribosome Binding Site Differentiation in Context Dependence

The ribosome binding site (RBS) is a crucial element regulating translation. However, the activity of RBS is poorly predictable, because it is strongly affected by the local possible secondary structure, that is, context dependence. By the Flowseq technique, over 20 000 RBS variants were sorted and sequenced, and the translation of multiple genes under the same RBS was quantitatively characterized to evaluate the context dependence of each RBS variant in E. coli. Two regions, (-7 to -2) and (-17 to -12), of RBS were predicted with a higher possibility to pair with each other to slow down the translation initiation. Associations between phenotypes and the intrinsic factors suspected to affect translation efficiency and context dependence of the RBS, including nucleotide bias at each position, free energy, and conservation, were disentangled. The results showed that translation efficiency was influenced more significantly by conservation of the SD region (-16 to -8), while an AC-rich spacer region (-7 to -1) was associated with low context dependence. We confirmed these characteristics using a series of synthesized RBSs. The average correlation between multiple reporters was significantly higher for RBSs with an AC-rich spacer (0.714) compared with a GU-rich spacer (0.286). Overall, we proposed general design criteria to improve programmability and minimize context dependence of RBS. The characteristics unraveled here can be adapted to other bacteria for fine-tuning target-gene expression.