(423c) 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. As operons tend to contain functionally related genes, they are thought to have evolved for regulatory purposes so that the genes contained in them will have similar transcriptional expression patterns. However, not all co-regulated genes reside in the same operon. Furthermore, alternative hypotheses have been proposed for the existence and prevalence for operons in bacteria, namely that they are the consequence of horizontal gene transfer. One aspect of operon structure is clear: operons link multiple genes to the same promoter and transcriptional regulatory sequences.

How operons affect translational regulation is still not known. On one hand, adjacent genes are often translationally coupled, where translation of the downstream gene is conditional on the translation of the upstream gene. Presumably, this mechanism is used to ensure that these genes are expressed at similar levels. On the other hand, the relative expression of adjacent genes can vary by orders of magnitude with no clear correlation between expression and ordering within the operon. Given these alternatives and everything in between, any modeling approach applied to translational regulation clearly needs to account for operon structure.

As a step towards developing such a model, 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 determined the factors that determine the degree of coupling between them. A mathematical model for translational coupling is proposed based on our experimental data.