## 3 Post-Transcriptional and Translational Regulation in Integrons
### The Role of attl1 Site on Integon-Associated Gene Cassette Translation
Once found in the second position, _aadB_ transcript amounts decreased in response to cassette position effect, but in a steady and linear fashion, different from what was observed when the level  
Figure 11: Sequence and organization of the _attII_ site and its regulatory translation signals ORF-11 and ORF-17. The gene cassette array of a class 1 integer is shown (personal communication). The SD sequences of ORF-11 and ORF-17 are in bold italicized caption letters. The start and stop codons of ORF-11 (red) and ORF-17 (blue) are highlighted in bold underlined caption letters. The start codon of the first gene cassette (_aacA7_) is also found in bold underlined caption letters. The 7-bp re-combination core site is boxed. The ORF-11 and ORF-17 overlapped coding sequences are fea-tured by red and blue horizontal arrows, respectively, and their predicted amino acid sequences are indicated. The asterisks represents the translation termination produced by ORF-11 (TAG) and ORF-17 (TAA) stop codons. of gentamycin resistance was considered. Such a remarkable difference in gentamycin resistance was explained by the fact that when _aadB_ was in the first integer position it was efficiently expressed by translation coupling with the ORF-11, reflecting in high levels of antibiotic resistance. Additionally, this study demonstrated that the several random mutations selected in response to antibiotic pressure led to a heightened translation efficiency of more distal gene cassettes either by incrementing their TIRs or by creating fused cassettes, which also favor translation coupling [124], as discussed below. Additionally to ORF-11, other ORFs can be created in detrimental of the features of the _attII_ site and inserted gene cassettes. It was observed that another ORF coding 18 aa, named ORF-18, was also involved with TIR-deficient gene cassette translation [125]. The TIR and the first 11 amino acids of ORF-18 were identical to those of ORF-11, but its stop codon was not provided by the recombination core site of the inserted cassette. In this case, the ORF-18 was created upon insertion of the _bla_OXA-1 gene cassette, which presents a 5'UTR of 66 bp. Since this cassette does not harbor a potential stop codon in its 7-bp core site, the open reading frame was enlarged in 21 bp into OXA-1 5'UTR (where an in-phase TAA stop codon was found), providing 7 additional amino acids to the short peptide. Therefore, the start codon of the OXA-1 gene was found 45 bp downstream of ORF-18, which contributed to gene cassette expression by the same translation coupling mechanism described for ORF-11 [122]. As found for ORF-11, ORF-18 deletion also abolished OXA-1 translation, which was reset upon the upstream insertion of an SD sequence [15]. The presence of ORF-18 was also detected upstream other TIR-deficient _bla_OXA-1-related gene cassettes, such as _bla_OXA-30 and _bla_OXA-31, and also in _anc(6)-Ib_ cassettes encoding acetyltransferaseases fused to the OXA-1 N-terminal end [15,125], demonstrated the intimate relationship between ORF-18 production and the presence of the OXA-1 5'UTR. Similarly, a third ORF of 51 bp, encoding a peptide of 17 aa (ORF-17) and harboring a proper TIR, was also intrinsically identified in the _attII_ site [123]. However, unlike ORF-11 and ORF-18, the ORF-17 production does not require the supply of a stop codon by the gene cassette inserted at the _attII_ site. Although ORF-17 overlaps ORF-11, they are encoded in different frames, in a way that ORF-17 starts 26 bp upstream and utilizes an in-frame stop codon inside ORF-11 (Figure 11). The ability of ORF-17 in enhancing translation of downstream gene cassettes was firstly assessed in a class 1 integer harboring the _bla_GES-1 inserted in the _attII_ site. However, in this case, _bla_GES-1 harbored its own canonical SD sequence. [PERSON] et al. [123] experimentally demonstrated that, in spite of the presence of a GES-1 SD sequence, the ORF-11 and ORF-17 were requested to reach highest gene cassette expression levels.