Quantitative analysis of translational coupling in Escherichia coli

Multiple genes are often transcribed on the same mRNA transcript in bacteria. These multi-gene transcriptional units, called operons, are widespread in bacteria with nearly half of all annotated genes predicted to reside in them. How operons affect translational regulation is still not known. There is a phenomenon specific to operon, so called translational coupling, which means that the translation of the downstream gene is often dependent upon the translation of the upstream gene. As a step towards developing a model for protein expression pattern in operon, we have engineered a synthetic two-gene operon, where translation of the downstream gene is conditional on the translation of the upstream gene. This operon encodes two fluorescent proteins, where the upstream gene is translated by orthogonal ribosomes and the downstream gene by native ribosomes. This design allows us to precisely tune the translation of the upstream gene and then record its affect on the translation of the downstream gene. Using this general system, we show that the translation of these two genes can be tightly coupled. We have also showed that the mRNA level is also tightly coupled with the gene expression level, which indicates the correlation between the amount of ribosome translating and the mRNA stability