Transcription regulation and termination in mycobacteria : DNA gyrase genes as a paradigm

Abstract

Molecular Biology of Mycobacterium tuberculosis: Transcriptional Regulation and DNA Gyrase Function

Abstract and Synopsis Introduction The textbook definition of prokaryotic molecular biology is largely based on experiments with Escherichia coli. Attempts to generalize findings to other bacteria often fail due to exceptions, which highlight the diversity and adaptability of prokaryotes. These exceptions complicate predictions about novel bacteria and hinder therapeutic development against infectious species. This thesis focuses on understanding the molecular biology of Mycobacterium tuberculosis-the leading cause of death worldwide-using DNA gyrase genes from M. tuberculosis and the non-pathogenic M. smegmatis as models.

Chapter 1 Introduces DNA topology, topoisomerases, and transcription. Reviews the discovery of DNA supercoiling, regulation of topoisomerases, and compares transcription in E. coli and mycobacteria. Discusses the impact of topology on transcription, with emphasis on virulence gene expression.

Chapter 2 Analyzes transcription start sites of gyrA and gyrB in M. smegmatis. Findings reveal supercoil-sensitive promoter regulation distinct from E. coli, and a nutrient-dependent regulation at the level of mRNA stability. This study also identifies dicistronic transcription of gyrB and gyrA-reported here for the first time.

Chapter 3 Characterizes promoters in the gyr locus of M. tuberculosis. While the major transcript is dicistronic, additional promoters (specific for gyrA and overlapping/divergent promoters) were identified. These promoters exhibit differential sensitivity to DNA topology.

Chapter 4 Examines autoregulation of DNA gyrase in E. coli. Theoretical analysis of promoter mutants revealed a strong bend center downstream of the -10 region of gyrA. Curvature correlated with supercoil sensitivity, leading to a model where axial distortion of DNA acts as a sensor for topology changes.

Chapter 5 Defines promoter elements in mycobacteria using random mutagenesis of the M. smegmatis gyr promoter. Identified elements crucial for promoter recognition by RNA polymerase, expanding understanding of transcription initiation in mycobacteria.

Chapter 6 Investigates transcription termination in M. tuberculosis. Unlike E. coli, G/C-rich mycobacteria lack classical U-trails. A novel algorithm was developed to identify secondary structures in bacterial genomes, revealing new classes of terminators. The refined algorithm successfully identified experimentally tested terminators across eubacteria.

Conclusions

Transcription initiation, regulation, and termination in mycobacteria differ significantly from E. coli.

Autoregulation of DNA gyrase is conserved across bacteria, but mechanisms vary by species.

Intrinsic transcription termination is conserved, but adaptations allow it to function in G/C-rich organisms without U-trails.

These findings contribute to a more accurate model of prokaryotic molecular biology, highlighting species-specific adaptations and evolutionary divergence shaped by functional selection.