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.

Sequence and thermodynamic characteristics of terminators revealed by FlowSeq and the discrimination of terminators strength.

The intrinsic terminator in prokaryotic forms secondary RNA structure and terminates the transcription. However, leaking transcription is common due to varied terminator strength. Besides of the representative hairpin and U-tract structure, detailed sequence and thermodynamic features of terminators were not completely clear, and the effect of terminator on the upstream gene expression was unclearly. Thus, it is still challenging to use terminator to control expression with higher precision. Here, in E. Coli, we firstly determined the effect of the 3'-end sequences including spacer sequences and terminator sequences on the expression of upstream and downstream genes. Secondly, terminator mutation library was constructed, and the thermodynamic and sequence features differing in the termination efficiency were analyzed using the FlowSeq technique. The result showed that under the regulation of terminators, a negative correlation was presented between the expression of upstream and downstream genes (r=-0.60), and the terminators with lower free energy corelated with higher upstream gene expression. Meanwhile, the terminator with longer stem length, more compact loop and perfect U-tract structure was benefit to the transcription termination. Finally, a terminator strength classification model was established, and the verification experiment based on 20 synthetic terminators indicated that the model can distinguish strong and weak terminators to certain extent. The results help to elucidate the role of terminators in gene expression, and the key factors identified are crucial for rational design of terminators, and the model provided a method for terminator strength prediction.

Fine-Tuning Multi-Gene Clusters via Well-Characterized Gene Expression Regulatory Elements: Case Study of the Arginine Synthesis Pathway in C. glutamicum.

Promoters and ribosome binding sites (RBSs) are routinely applied in gene expression regulation, but their orthogonality and combinatorial effects have not yet been systematically studied in Corynebacterium glutamicum. Here, 17 core promoters and 29 RBSs in C. glutamicum were characterized, which exhibited 470-fold and 430-fold in transcriptional and translational activity, respectively. By comparing the expression of two reporter genes regulated by multiple RBSs, the RBS efficacy showed significant dependence on the gene context, besides the RBSs' strength, reflecting the poor orthogonality of RBSs. Bicistron-modified RBS (referred as bc-RBS) was adapted to C. glutamicum, which improved RBS reliability. By coupling a series of promoters with RBSs/bc-RBSs, a much broader regulation range that spanned 4 orders of magnitude was observed compared with that of a sole element, and the contribution to gene expression of RBS was more than that of promoter. Finally, promoters and RBSs were applied as built-in elements to fine-tune the gene cluster in the arginine synthesis pathway in C. glutamicum. Compared with the original strain, more arginine (1.61-fold) or citrulline (2.35-fold) was accumulated in a 7 L bioreactor by strains with the gene expression regulation system rationally engineered. We demonstrated that, via combination of well-characterized gene elements, and overall consideration for both transcription and translation, the biosynthesis pathway can be effectively balanced, and the yield of a target metabolite can be further improved.

Evaluating Terminator Strength Based on Differentiating Effects on Transcription and Translation.

Transcription terminators play a role in terminating the progress of gene transcription, and are thus essential elements in the gene circuit. Terminators have two main functions: terminating gene transcription and improving the stability of gene transcripts during translation. We therefore considered the detailed characteristics of terminators in relation to their different roles in gene transcription and translation, including transcription shut-down degree (α) and upstream mRNA protection capacity (ß), and apparent termination efficiency (η) reflecting the overall regulatory effect of the terminator. Based on a dual-reporter gene system, we constructed three terminator-probe plasmids to investigate each characteristic in Escherichia coli. According to multiple regression analysis, the transcription shut-down degree and the upstream mRNA protection capacity contributed almost equally to the apparent termination efficiency. Sequence analysis of 12 terminators demonstrated that the terminator sequence was dominated by GC bases, and that a high ratio of GC bases in the stem structure of terminators might be associated with a high degree of transcription shut-down. This comprehensive characterization of terminators furthers our understanding of the role of terminators in gene expression and provides a guide for synthetic terminator design.