Oligomerization states of the Mycobacterium tuberculosis RNA polymerase core and holoenzymes

Arch Microbiol. 2024 Apr 22;206(5):230. doi: 10.1007/s00203-024-03955-z.

Abstract

During the past few decades, a wealth of knowledge has been made available for the transcription machinery in bacteria from the structural, functional and mechanistic point of view. However, comparatively little is known about the homooligomerization of the multisubunit M. tuberculosis RNA polymerase (RNAP) enzyme and its functional relevance. While E. coli RNAP has been extensively studied, many aspects of RNAP of the deadly pathogenic M. tuberculosis are still unclear. We used biophysical and biochemical methods to study the oligomerization states of the core and holoenzymes of M. tuberculosis RNAP. By size exclusion chromatography and negative staining Transmission Electron Microscopy (TEM) studies and quantitative analysis of the TEM images, we demonstrate that the in vivo reconstituted RNAP core enzyme (α2ββ'ω) can also exist as dimers in vitro. Using similar methods, we also show that the holoenzyme (core + σA) does not dimerize in vitro and exist mostly as monomers. It is tempting to suggest that the oligomeric changes that we see in presence of σA factor might have functional relevance in the cellular process. Although reported previously in E. coli, to our knowledge we report here for the first time the study of oligomeric nature of M. tuberculosis RNAP in presence and absence of σA factor.

Keywords: M. tuberculosis RNA polymerase; Holoenzyme; Negative staining TEM; Oligomerization; Transcription.

MeSH terms

  • Bacterial Proteins* / chemistry
  • Bacterial Proteins* / genetics
  • Bacterial Proteins* / metabolism
  • Chromatography, Gel
  • DNA-Directed RNA Polymerases* / chemistry
  • DNA-Directed RNA Polymerases* / genetics
  • DNA-Directed RNA Polymerases* / metabolism
  • Holoenzymes / chemistry
  • Holoenzymes / metabolism
  • Microscopy, Electron, Transmission
  • Mycobacterium tuberculosis* / chemistry
  • Mycobacterium tuberculosis* / enzymology
  • Mycobacterium tuberculosis* / genetics
  • Protein Multimerization*
  • Sigma Factor / chemistry
  • Sigma Factor / genetics
  • Sigma Factor / metabolism

Substances

  • DNA-Directed RNA Polymerases
  • Holoenzymes
  • Bacterial Proteins
  • Sigma Factor