RESUMO
The transcription factor Rv0081 of Mycobacterium tuberculosis controls hypoxic gene expression and acts as a regulatory hub in the latent phase of tuberculosis (TB) infection. We report here the crystal structure of Rv0081 at 2.9 Å resolution revealing that it belongs to the well-known ArsR/SmtB family proteins. However, unlike other members in this family, Rv0081 has neither a metal-binding domain nor does it possess Cys residues, suggesting an alternate mechanism of gene regulation. Our structural and biochemical analyses suggest the molecular basis for the recognition of self-regulatory DNA sequences and a plausible mechanism of regulation of Rv0081 in the latent phase of TB infection. DATABASE: Structural data are available in the Protein Data Bank under the accession number - 6JMI.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/genética , Oxigênio/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , DNA Bacteriano/metabolismo , Cinética , Modelos Moleculares , Mutação , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Fatores de Transcrição/genéticaRESUMO
The ability of Mycobacterium tuberculosis (M. tb) to survive and persist in the host for decades in an asymptomatic state is an important aspect of tuberculosis pathogenesis. Although adaptation to hypoxia is thought to play a prominent role underlying M. tb persistence, how the bacteria achieve this goal is largely unknown. Rv0081, a member of the DosR regulon, is induced at the early stage of hypoxia while Rv3334 is one of the enduring hypoxic response genes. In this study, we uncovered genetic interactions between these two transcription factors. RNA-seq analysis of ΔRv0081 and ΔRv3334 revealed that the gene expression profiles of these two mutants were highly similar. We also found that under hypoxia, Rv0081 positively regulated the expression of Rv3334 while Rv3334 repressed transcription of Rv0081. In addition, we demonstrated that Rv0081 formed dimer and bound to the promoter region of Rv3334. Taken together, these data suggest that Rv0081 and Rv3334 work in the same regulatory pathway and that Rv3334 functions immediately downstream of Rv0081. We also found that Rv3334 is a bona fide regulator of the enduring hypoxic response genes. Our study has uncovered a regulatory pathway that connects the early and the enduring hypoxic response, revealing a transcriptional cascade that coordinates the temporal response of M. tb to hypoxia.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/genética , Proteínas Quinases/genética , Fatores de Transcrição/genética , Anaerobiose/genética , Proteínas de Ligação a DNA , Mycobacterium tuberculosis/patogenicidade , Oxigênio , Análise de Sequência de RNA , Tuberculose/microbiologiaRESUMO
Because of its high infectivity and pathogenicity, Mycobacterium tuberculosis is a serious threat to human health. While the transcription-regulatory system of M.â tuberculosis remains incompletely understood, Rv0081, an essential regulatory hub, is known to mediate the initial response to hypoxia in the long-term survival of M. tuberculosis. Here, the production, crystallization and initial X-ray crystallographic analysis of Rv0081 are reported. The crystals of Rv0081 belonged to space group P62, with unit-cell parameters a = 67.48, b = 67.48, c = 40.84â Å, γ = 120°. The Matthews coefficient is 2.09â Å3â Da-1, assuming the presence of one molecule in the asymmetric unit, with a corresponding solvent content of 41.27%. Phasing of the native crystal form of Rv0081 was performed by molecular replacement. Currently, the structure has been refined to 2.00â Å resolution with an Rwork of 25.99% and an Rfree of 30.88%.