## 3 Post-Transcriptional and Translational Regulation in Integrons
### The Role of attl1 Site on Integon-Associated Gene Cassette Translation  
The presence of a ribosomal binding site (RBS), known as the Shine-Dalgarno sequence in bacteria (SD sequence), is crucial for the recruitment and assembly of ribosomes and initiation of gene translation. A proper SD sequence usually comprises four bases, normally GGAG, which is complementary to the 165 rRNA small subunit [121]. The efficient expression of gene cassettes inserted in integrons depends on the presence of the key elements of a canonical translation initiation region (TIR), represented by a functional SD sequence, a plausible start codon, and a proper spacer between them (about 8 bp). Although most of the gene cassettes present a canonical TIR, about 20% of them are devoid of these translational components. In this case, such TIR-deficient gene cassettes may benefit from some regulatory signals found in the _attl1_ site when inserted in the first position of class 1 integrins. A small ORF coding 11 amino acids (aa), named ORF-11, is able to enhance the gene expression at the translational level by recruiting ribosomes to TIR-deficient cassettes. The ORF-11 has a canonical TIR, composed of a functional SD sequence (GGAG) placed 8 bp upstream of the ATG start codon. This ORF spans the _attl1_ site of class 1 integrins, placed 2-62 bp upstream of the first inserted gene cassette. Since ORF-11 is intrinsically encoded in the _attl1_ site, it is present in all transcripts originating from the Pc [122]. However, the ORF-11 overlaps the recombination crossover point of the _attII_ site, in a way that the production of the 11-aa peptide depends on the supply of an in phase stop codon by the first cassette inserted in the array. This stop codon (usually TAG) lies on positions 3 to 5 of the 7 bp GTTRRRY _attC_ core site of the inserted cassette (Figure 11). In this way, after ORF-11 translation, the terminated ribosomes might not be released at its stop codon, which leads to a translation restart at initiation codon of the TIR-deficient gene cassette by lateral diffusion of ribosomes through the transcript. Therefore, the translation of ORF-11 supports the expression of the first gene cassette by a translational coupling mechanism, which occurs in polycistronic transcripts where the termination and start codons of contiguous genes are overlapping or close to each other. In fact, the inspection of published sequences supports that the expression of a great proportion of gene cassettes inserted at _attII_ may benefit from translational coupling with ORF-11. [PERSON] et al. [122] also demonstrated that ORF-11 deletion and replacement of its SD sequence or ATG start codon with non-canonical sequences dramatically decreased its translation efficiency. On the other hand, amino acid substitutions in ORF-11 had only a slight effect on translation. Those results demonstrated that the translated ORF-11 itself, and especially its TIR, plays a significant role in enhancing the expression of several TIR-deficient gene cassettes. However, not only TIR-deficient cassettes can be found fused to ORF-11. A remarkable proportion of cassettes with a proper TIR have been found inserted in the first position of integrins coding functional ORF-11 in their _attII_ sites [123]. In this case, an enhanced translation may take place, since both TIRs could augment the recruitment of ribosomes in the vicinity of the gene cassette initiation codon. A recent study experimentally evaluated the \"evolution on demand\" of antibiotic resistance in response to selective pressures and integrase activity, and the important role of ORF-11 on antibiotic resistance manifestation was established in that context [124]. It was demonstrated that when the TIR-deficient _aadB_ gene cassette was placed in the second position on the integrin array, the levels of gentamycin resistance suffered a drastic reduction relative to when the _aadB_ was found in the first position. However, this significant difference was not verified when their transcript amounts were evaluated.