Sequence polymorphisms in G-quadruplex region of Mycobacterium tuberculosis espK affect gene expression
HAN Angxuan, NIU Chen, LU Liangdong
Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
Abstract：Four-stranded helical structures called G-quadruplexes (G4s) formed by G-rich DNA sequences are non-canonical structures. G4s are now known to affect gene expression and genome stability. Studies have suggested that Mycobacterium tuberculosis (M. tuberculosis) espK is an important component of ESX-1 (ESAT-6 secretion system-1), and its protein sequence has tandem repetitive GTPITP sequence polymorphism. In this study, we identified a G4 sequence of espK (Rv3879c) on the template strand corresponding to GTPITP sequence polymorphism region, and this G4 sequence only existed in M. tuberculosis complex (MTBC). By comparing espK gene nucleic acid sequence in the clinical M. tuberculosis isolates, we confirmed a high-frequency G4 single nucleotide polymorphism (SNP), G1573C mutation in espK gene. The purpose of the study is to analyze the structure and expression regulation of espK G4. Firstly, espK G4 nucleic acid fragment in vitro could form parallel G4 structure in the presence of K＋ by circular dichroism spectrum detection and the structural stability of G4 decreased after synonymous point mutation of G4. We constructed espK expression plasmid containing synonymous point mutant G4 by overlapping polymerase chain reaction (PCR), and further constructed recombinant strains with espK gene of synonymous point mutant G4 and wild-type espK. By quantitative real-time PCR detection, transcription level of recombinant strains with espK gene of synonymous point mutant G4 was significantly upregulated (P<0.05). At the same time, the high-frequency G1573C mutant in espK could destabilize G4 structure and resulted in increased protein expression level in the recombinant strain by Western blotting. It is thus concluded that G4 of M. tuberculosis espK could regulate expression, and the sequence polymorphism in espK G4 region may regulate the activity of ESX-1 secretion system through affecting the expression level of EspK.
韩昂轩，牛辰，吕亮东. 结核分枝杆菌espK基因G-四链体核酸序列多态性与表达调控功能研究[J]. 微生物与感染, 2018, 13(2): 77-83.
HAN Angxuan, NIU Chen, LU Liangdong. Sequence polymorphisms in G-quadruplex region of Mycobacterium tuberculosis espK affect gene expression. JOURNAL OF MICROBES AND INFECTIONS, 2018, 13(2): 77-83.
2018, Vol. 13 
