page_title: Αr15 RNA
section_title: Promoter analysis
hierarchical_section_title: Αr15 RNA / Promoter analysis
caption_attribution_description: English: 15C promoters
context_page_description: αr15 is a family of bacterial small non-coding RNAs with representatives in a broad group of α-proteobacteria from the order Rhizobiales. The first members of this family were found tandemly arranged in the same intergenic region of the Sinorhizobium meliloti 1021 chromosome. Further homology and structure conservation analysis have identified full-length Smr15C1 and Smr15C2 homologs in several nitrogen-fixing symbiotic rhizobia, in the plant pathogens belonging to Agrobacterium species as well as in a broad spectrum of Brucella species. The Smr15C1 and Smr15C2 homologs are also encoded in tandem within the same IGR region of Rhizobium and Agrobacterium species, whereas in Brucella species the αr15C loci are spread in the IGRs of Chromosome I. Moreover, this analysis also identified a third αr15 loci in extrachromosomal replicons of the mentioned nitrogen-fixing α-proteobacteria and in the Chromosome II of Brucella species. αr15 RNA species are 99-121 nt long and share a well defined common secondary structure consisting of three stem loops.
context_section_description: All αr15 loci have recognizable σ⁷⁰-dependent promoters showing a -35/-10 consensus motif CTTGAC-n17-CTATAT, which has been previously shown to be widely conserved among several other genera in the α-subgroup of proteobacteria. A multiple sequence alignment of these promoter regions revealed a conserved sequence stretch extending up to 80 bp upstream of the transcription start site in all the αr15 loci with the only exceptions of the S. meliloti, S.fredii and S. medicae αr15C1 promoters.
  To identify binding sites for other known transcription factors we used the fasta sequences provided by RegPredict(http://regpredict.lbl.gov/regpredict/help.html), and used those position weight matrices (PSWM) provided by RegulonDB  (http://regulondb.ccg.unam.mx). We built PSWM for each transcription factor from the RegPredict sequences using the Consensus/Patser program, choosing the best final matrix for motif lengths between 14–30 bps a threshold average E-value < 10E-10 for each matrix was established, (see "Thresholded consensus" in http://gps-tools2.its.yale.edu). Moreover, we searched for conserved unknown motifs using MEME (http://meme.sdsc.edu/meme4_6_1/intro.html) and used relaxed regular expressions (i.e. pattern matching) over all αr15 homologs promoters.
  These studies revealed a difference in regulation between S. melilloti, S.fredii and S. medicae αr15C1 sRNAs and the rest of the αr15 family members independently of their group of origin (Rhizobium or Agrobacterium ) and genomic location (αr15C1, αr15C2, αr15plasmid) (Figure 4).
  The rest of the family members presented a very well conserved 30 bp long region between positions -36 and -75. This conserved region, was used to query databases of known transcription factor motifs with TomTom, the best matching motif was SMb20667_Rhizobiales, motif belonging to RegTransBase (http://regtransbase.lbl.gov/cgi-bin/regtransbase?page=main). The Smb20667 correspond to the binding site for a dicarboxylate metabolism regulator in Rhizobiales that belongs to the LacI family. This motif was identified clustering genes of tartrate dehydrogenase, succinate semialdehyde dehydrogenase, 3-hydroxyisobutyrate dehydrogenase and hydroxypyruvate isomerase in S. meliloti, and several Rhizobiums  and it is marked in the promoter alignment figure  (Figure 5) with an orange box. Moreover, another conserved sequence was found using MEME in the promoter region of the Sinorhizobium species αr15C1. This conserved region was used to query databases of known transcription factor motifs with TomTom, and the best matching motif was SMb20537_Rhizobiales (Figure 5 in red), that identifies the binding site for a sugar utilization regulator in Rhizobiales from the LacI family as well.
caption: Figure 5: Graphic representation of the 15C seed members' promoter region. All members presented putative σ70 promoters with -30 and -10 boxes marked in green and red respectively. Conserved motifs are marked with orange and red color boxes.
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