Translational coupling via termination-reinitiation in archaea and bacteria

Translational coupling occurs at many gene pairs of archaea and bacteria. Translational coupling means that translation of the downstream gene on a polycistronic transcript strictly depends on translation of the upstream gene. One specific mechanism of coupling is called termination-reinitiation (TeRe). In this case, the translating ribosome (at least the small subunit) remains on the mRNA after terminating translation of the upstream gene, and it directly continues translation by reinitiating at the downstream gene. This mechanism typically occurs at gene pairs that have overlapping stop/start codons or very small intergenic distances.

In previous studies we have shown that TeRe operates at several native gene pairs of Haloferax volcanii and Escherichia coli¹ and that efficient coupling required very short intergenic distances². Translational efficiencies were quantified using reporter genes fused to native gene pairs, enzyme assays and northern blotting were used to determine protein and transcript levels. In the present study, we investigated whether local mRNA structures at gene overlaps in E. coli are required to inhibit novel initiation at the downstream genes and guarantee coupling. To this end, we generated several constructs with truncated or mutated gene pairs, with the aim of destabilizing local mRNA structures close to the overlaps. The effects of these genetic perturbations on the efficiencies of translation and coupling will be reported.

Additionally we created a random library with a 42 random nucleotide sequence directly upstream of the overlapping start/stop codon. In our two-reporter gene system, this library is used to select and screen for sequence motives that are important for efficient novel initiation at the downstream gene as well as translational coupling via TeRe.

Taken together, translational coupling via TeRe operates at many gene pairs in archaea and bacteria, and various aspects of the molecular mechanism could be characterized using native gene pairs fused to reporter genes.

¹ Huber et al. (2019) Nature Comm. 10: 4006.

² Huber, Vogel et al. (2023) Front Microbiol. 14: 1291523.