Characterization and implications of prokaryotic ribosome-binding sites across species

The ribosome-binding site (RBS) in the 5′ untranslated region is recognized by 16S rRNA to start translation and is an essential element of the gene expression system. RBSs have been widely applied in regulating gene expression in various scenarios, including Gram-negative or Gram-positive bacteria. Here, we first rationally designed and constructed an RBS mutant library containing 66 RBSs. The strength of these RBSs in E. coliand C. glutamicumwas characterized individually. The RBS strength spanned about 200 and 15 times in the two species, respectively. The strength of RBSs in C. glutamicumwas generally lower than that of in E. coli. A total of 18 RBSs showed similar strength (within twofold differences) between the species in our study, and the correlation analysis of the strength of RBSs between E. coliand C. glutamicum(R2= 0.7483) revealed that these RBSs can be used across species. The sequence analysis revealed that the RBS region with two Ts stated was beneficial for RBS to function cross-species. The RBS characterized here can be used to precisely regulate gene expression in both hosts, and the characteristics of cross-species RBSs provide basic information for RBS rational design.