Deciphering the Rules of Ribosome Binding Site Differentiation in Context Dependence

Abstract

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.

Flow-Seq Evaluation of Translation Driven by a Set of Natural Escherichia coli 5′-UTR of Variable Length

“…In the current work, we assessed the influence of 5′-UTR on translation in E. coli while using a set of natural 5′-untranslated regions with lengths in the range of 2–60 nt. The obtained results fit the expectations and earlier experiment results with randomized 5′-UTR sequences well [ 6 , 7 , 16 ]. We observed an influence in secondary structures [ 21 ] and Shine–Dalgarno sequences on translation, although the variability of these parameters in the current dataset was much smaller in comparison with those in randomized 5′-UTR libraries.…”

“…This could be explained by the fact that we used natural 5′-UTRs that universally possess optimal necessary ribosome binding elements, while translation efficiency is adjusted by other means, such as, e.g., a codon frequency adjustment. Likewise, we could not see a clear difference in the AC nucleotide content between the fractions as was suggested earlier on the basis of a randomized 5′-UTR analysis [ 16 ].…”

“…Flow-seq is the method of choice for studying sequence elements that influence gene expression at all levels. This method allows one to test a wealth of natural [ 20 ] as well as artificial [ 6 , 16 ] regulatory elements in a standardized environment. In natural genes, all factors influence expression simultaneously, so it can be difficult to decipher the influence of any single factor.…”

Flow-Seq Method: Features and Application in Bacterial Translation Studies

“…A number of studies employing the Flow-seq method have investigated the effect of the sequences of 5’ untranslated regions of different lengths and their individual sites on the efficiency of the reporter fluorescent protein synthesis [ 2 , 39 , 71 , 72 , 73 , 74 ].…”

“…The Flow-seq method has been repeatedly used to gain insight into how the nucleotide sequences of different motifs of 5’ UTRs affect the translation efficiency. In particular, the ribosome binding sites with a fixed SD sequence [ 74 ], 5’ UTRs of different fixed lengths [ 2 ], or natural 5’ UTRs of different lengths [ 77 ], as well as standby sites and spacer regions [ 72 ], were studied. An analysis of tens of thousands of tested variants showed that the variation in the efficiency of the reporter protein synthesis can amount to four, and even five, orders of magnitude.…”