Functional characterization of G-quadruplexes in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is the etiologic agent of tuberculosis, the second cause of death from a single infectious agent after COVID-19. In the last decades, the emergence of Mtb drugresistant strains is further pushing research towards the discovery of new therapeutic targets. Gquadruplexes (G4s) are non-canonical secondary DNA structures that form in guanine-rich sequences and are involved in several fundamental biological processes. We have previously identified putative G4s in Mtb genome and showed that G4-ligands such as BRACO-19 (B19) and c-exNDI are able to inhibit bacterial growth. One of our main goals is to discover the biological function of G4s in Mtb. To this aim, we performed an RNA-seq experiment under treatment with the G4-ligand (B19). Data analysis revealed a correlation between the presence of putative G4s (pG4s) in the coding regions of the differentially expressed genes and their level of expression, suggesting a relationship between G4s and gene expression. We proved that the presence of pG4s in the coding regions of genes decreases gene transcripts decay rate, providing a mechanism that may contribute to the cellular mRNA pool. We also investigated the role of a pG4 in the expression regulation of lpqS, and proved by EMSA that B19 treatment inhibits RicR repressor binding. This study is one of the first in vivo studies on the function of G4s in bacterial cells. It proves that G4s may be involved in gene expression regulation both at a pre- and post- transcription level. It gives many relevant insights that will guide future deeper studies.