Translation regulation of integrons gene cassette expression by the attC sites

P>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')-Ib(7) gene. In the resulting constructs, the 5'-attC site differentially affected the expression of the aac(6')-Ib(7) gene. Contrary to what was expected from their proposed role as Rho-independent transcription terminators, the transcription of the aac(6')-Ib(7) 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.