Translation regulation of integrons gene cassette expression by the attC sites

Integron are genetic elements able to carry, capture and shuffle the genes embedded in gene cassettes. The attC recombination sites adopt a stable secondary structure when single-stranded that is necessary for their recombination. In this study, we evaluated the impact of the structure of the attC site on expression of the 3' gene in class 1 integrons. This was analysed by substituting the attC of the bla(IMP-8) gene cassette with various mutated attC sites spanning a wide range of sizes and secondary structures, and measuring the integron-dependent translation of the 3'aac(6')-Ib7 gene. In the resulting constructs, the 5'-attC site differentially affected the expression of the aac(6')-Ib7 gene. Contrary to what was expected from their proposed role as Rho-independent transcription terminators, the transcription of the aac(6')-Ib7 gene was not affected by the various attC sites. Mutations of natural sites revealed that destabilization of the potential stem-loop structure of the attC site in the transcript could enhance the expression of the 3' gene. In particular, the presence of a translated open reading frame was shown to increase translation of the 3' gene. These findings might be explained by the capacity of the stem-loop structures to impede ribosome progression.