## 2 Transcriptional Regulation in Integrons
### The Role of Pc Promoters on Gene Cassette Transcription
#### 2.1.2 Pc Promoter in Class 2 Integrons  
The class 2 integor Pc promoter sequence was precisely determined and functionally characterized in studies that used different approaches. These platforms are associated with transposons related to Tn7 and usually carry _dfrA1_, _sat2_, and _aadA1_ resistance gene cassettes followed by the pseudo-cassette _ybeA_ of unknown function [45]. Variations in this gene cassette array are rarely reported due to truncation of the _intl2_ gene by a premature ochre stop codon (TAA) at position 179, which yielded an inactive shorter protein [46]. Previous studies had pointed to the presence of four potential class 2 Pc promoters (Pc2A to Pc2D) located within the _attl12_ region [47; 48; 46]. However, the configuration and activity of any of these Pc2 have not been assessed in these studies. Subsequently, a fifth putative Pc2 promoter, Pc2E, was found embedded in the _intl2_ coding sequence displaying a TGN-10 motif [48] (Table 3). [PERSON] and Colleagues [49] determined the exact sequence and the activity of a Pc2 promoter by performing in silico promoter prediction analysis and in vitro characterization of the transcription start site (TSS) of the gene cassette array in class 2 integorns (Figure 2). It was demonstrated that this Pc, which corresponded to the previously mapped Pc2A, was responsible for the production of gene cassette transcripts and antibiotic resistance manifestation. This Pc2A (Table 3) resembled \(\sigma^{70}\)-dependent promoters, preserving 8 of 12 nucleotides of the consensus sequence -35 TTGACA (16-18 bp) -10 TATAAT. Different from class 1 Pc promoters, which are found embedded in the _intl1_ gene [3], the Pc2A was embedded in the _attl2_ site, 192 bp upstream _dfrA1_, the first gene cassette usually found in class 2 integrins [45; 46]. These findings corroborated previous studies that suggested the existence of a possible promoter region in that recombination site [47; 46]. Moreover, the Pc2A was found immediately upstream (five nucleotides) from the characterized TSS of gene cassette array and taking into account that the distance between a promoter and the TSS is usually 7-10 nucleotides, this Pc was assigned to be responsible for transcription of the resistance gene cassettes (Figure 2) [49]. Considering the sequence of the four major class 1 integor Pc promoter variants (PcS, PcW, PcH1, and PcH2), the Pc2A was more similar to PcS, presenting three and one polymorphisms relative to -35 and -10 hexamers, respectively. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline Pc2 & & & & & & \\ Promoter & \text  
Another study addressing this issue [48] experimentally determined the functionality and the relative transcription strength of the five Pc2 promoters, Pc2A to Pc2E (Figure 3). It was demonstrated that the Pc2A, already characterized in [PERSON] et al. [49], Pc2B and Pc2C were functional and that the formers have an equal contribution to gene cassette expression, while Pc2C presented a negligible transcription activity (Table 3). This simultaneous occurrence of different active Pc2 promoters is a unique feature of class 2 integrins among RIs/MIs, which could be involved to the differential activation of Pc2A and/or Pc2B under particular environmental conditions [48]. The in silico analysis of hundreds of class 2 integro sequences revealed, in rare cases, the occurrence of polymorphisms in Pc2A and Pc2B promoters, indicating the presence of class 2 Pc variants that could influence promoter strength and antibiotic resistance, as previously noticed for class 1 Pc promoters [26]. These Pc2 variants presented an A/Gmutation at the Pc2A -10 hexamer (TAAAGT), and a G/A mutation at Pc2B -35 hexamer (TTATAT), and these variants were named Pc2A-V2 and Pc2B-V2, respectively (Table 3). These rare Pc2 variants were less efficient in transcribing inserted gene cassettes than the canonical Pc2A and Pc2B, and they occurred only when the intact and active \(intl2\) gene, without the ochre premature stop codon, was present [48; 50; 51; 52; 53].