CarD stabilizes mycobacterial open complexes via a two-tiered kinetic mechanism

Nucleic Acids Res. 2015 Mar 31;43(6):3272-85. doi: 10.1093/nar/gkv078. Epub 2015 Feb 19.

Abstract

CarD is an essential and global transcriptional regulator in mycobacteria. While its biological role is unclear, CarD functions by interacting directly with RNA polymerase (RNAP) holoenzyme promoter complexes. Here, using a fluorescent reporter of open complex, we quantitate RPo formation in real time and show that Mycobacterium tuberculosis CarD has a dramatic effect on the energetics of RNAP bound complexes on the M. tuberculosis rrnAP3 ribosomal RNA promoter. The data reveal that Mycobacterium bovis RNAP exhibits an unstable RPo that is stabilized by CarD and suggest that CarD uses a two-tiered, concentration-dependent mechanism by associating with open and closed complexes with different affinities. Specifically, the kinetics of open-complex formation can be explained by a model where, at saturating concentrations of CarD, the rate of bubble collapse is slowed and the rate of opening is accelerated. The kinetics and open-complex stabilities of CarD mutants further clarify the roles played by the key residues W85, K90 and R25 previously shown to affect CarD-dependent gene regulation in vivo. In contrast to M. bovis RNAP, Escherichia coli RNAP efficiently forms RPo on rrnAP3, suggesting an important difference between the polymerases themselves and highlighting how transcriptional machinery can vary across bacterial genera.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • DNA, Bacterial / genetics
  • DNA-Directed RNA Polymerases / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Kinetics
  • Models, Biological
  • Mutagenesis, Site-Directed
  • Mycobacterium / genetics
  • Mycobacterium / metabolism*
  • Mycobacterium bovis / genetics
  • Mycobacterium bovis / metabolism
  • Mycobacterium tuberculosis / genetics
  • Mycobacterium tuberculosis / metabolism
  • Promoter Regions, Genetic
  • RNA, Bacterial / genetics
  • RNA, Ribosomal / genetics
  • Thermodynamics
  • Transcription Factors / chemistry
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Transcription Initiation, Genetic

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • Escherichia coli Proteins
  • RNA, Bacterial
  • RNA, Ribosomal
  • Transcription Factors
  • DNA-Directed RNA Polymerases