The genomic SELEX-based method identifies 350 SigA-specific promoters in Mycobacterium tuberculosis

The gene regulation in Mycobacterium tuberculosis by different sigma factors, including the principal sigma factor, sigmaA (SigA), is poorly understood. Here, we have developed a modified genomic systematic evolution of ligands by exponential enrichment (SELEX)-Seq approach that identifies 350 new SigA-binding sites in M. tuberculosis. SigA-binding ability and promoter activity of representative DNA sequences were confirmed by electrophoretic mobility shift assay (EMSA) and reporter assay, respectively. Among these DNA sequences, 38 are located in the intergenic region, indicating these regions as possible SigA promoters of the surrounding genes. The remaining 312 DNA sequences are located within the intragenic region, suggesting a previously unknown role of these binding sites, including SigA-dependent regulatory roles. We reveal that the intragenic SigA-binding sites are responsible for synthesizing 62 transcripts and 14 noncoding RNAs from the existing database. We have further identified 88 new proteins, different from annotated open reading frames (ORFs) in the genome sequences, downstream of the intragenic SigA-binding sites. Out of 350 SigA-binding sites, (a) 156 sequences contain -10 elements (T[C][N][N]N[T]) with a certain degree of degeneracy, including 38 having an additional extended -10 TG sequence, (b) 66 DNA sequences contain both -35 (T[G/T][G/T][C/T][N][C]) and -10 elements with a spacer of 5-25 bp, and (c) intriguingly, 128 SigA-binding sites contain only 35-like elements. Thus, our study reveals that the promoter architecture of M. tuberculosis significantly differs from the generalized concept of bacterial promoters and opens a new avenue to study gene regulation in M. tuberculosis.