## 2 Transcriptional Regulation in Integrons
### The Role of Cassette-Borne Promoter on Gene Cassette Transcription  
As discussed above, the transcription of gene cassettes placed in distal positions in an integron array decreases in response to their physical distance relative to the Pc promoter. However, in spite of the general promoterless nature of gene cassettes, some of these structures may harbor longer \(5^{\prime}\) untranslated regions (UTRs) that afford the presence of their own specific promoters [47]. The presence of such cassette-borne promoters breaks down the dogma of the cassette position effect, ensuring constitutive expression regardless of Pc configuration and gene cassette position within the array (Figure 4). So far, internal cassette promoters have been described in a diversity of gene cassettes involved with both resistance and non-resistance functions, as addressed below. Figure 4: Schematic representation of a class 1 integrin with additional gene cassette promoter regions. The Pc, P\({}_{\text{OUT}}\) (supplied at the \(3^{\prime}\) end of the insertion sequence), and the cassette-borne promoters P\({}_{\text{cmIAS}}\) and P\({}_{\text{OXA-10}}\) are represented by broken arrows. The influence of cassette-borne promoters in transcription efficiency is represented by black lines. The _cmLA1_ was the first identified gene cassette containing its own promoter. This promoter (P\({}_{\text{cmLA}}\)) showed to be functional as determined by promoter-probe vector assays expressing the _cat_ marker gene, and the high chloramphenicol resistance levels. P\({}_{\text{cmLA}}\) was found in the 5'UTR of _cmLA1_ cassette, 145 bp upstream its start codon. Afterward, the characterization of other _cmLA_ alleles revealed that this gene family was featured by carrying their own promoters. It was demonstrated that the P\({}_{\text{cmLA}}\) was conserved in the _cmLA2_ allele, differing by two polymorphisms in the -35 region, while the _cmLA5_ allele harbored the canonical P\({}_{\text{cmLA}}\)[61; 62; 63; 41]. In spite of being in the seventh position in the In53 integration array, the _cmLA5_ was efficiently transcribed by its active internal promoter (Figure 4) and conferred chloramphenicol resistance, even in distal positions relative to the Pc promoter [41]. The _qac_ resistance gene cassettes also carry internal promoters. Both _qacE_ and _qac1_ present long 5'UTR sequences containing a promoter region that seemed to be active in expressing them, leading to quaternary ammonium compound resistance [2; 41; 64]. Interestingly, a 101-bp segment (qacE101) of the _qacE_ 5'UTR has been found fused to the 5'end of several intergon-associated gene cassettes, such as _aac(6')-lb-cr_, _aacA29a_, _aacA29b_, and _aadA16_, which were transcribed by the _qacE_ promoter embedded in this segment [64; 65; 66; 67; 68]. Therefore, the qacE101 segment consists of a pseudo-cassette acting on transcription of downstream promoterless cassettes, representing an additional strategy to counterbalance the expression level reduction in the integron cassette arrays and/or the weakness of the Pc promoter. The presence of a cassette-borne promoter has also been identified in the context of class 2 integrins. [PERSON] and [PERSON] [47] demonstrated that the _ereA_ gene cassette, placed in the second position of a class 2 intergon array, harbored a functional promoter in its 5'UTR, placed 21 bp upstream the coding region. Most of these cassette-borne promoters reported to date had been characterized by means of in silico analysis and the indirect observation of antibiotic resistance phenotype in controlled systems [62; 63; 41; 47]. [PERSON] et al. [69] performed in silico, several in vitro and in vivo analyses to fully characterize an internal promoter found in the atypical _qnrVC1_ gene cassette, involved with decreased susceptibility to quinolones [10]. The _qnrVC1_ was the second cassette in a class 1 integer array, preceded by _aadA2_.