title: Effects of Increasing the Affinity of CarD for RNA Polymerase on Mycobacterium tuberculosis Growth, rRNA Transcription, and Virulence.
abstract: CarD is an essential RNA polymerase (RNAP) interacting protein in Mycobacterium tuberculosis that stimulates formation of RNAP-promoter open complexes. CarD plays a complex role in M. tuberculosis growth and virulence that is not fully understood. Therefore, to gain further insight into the role of CarD in M. tuberculosis growth and virulence, we determined the effect of increasing the affinity of CarD for RNAP. Using site-directed mutagenesis guided by crystal structures of CarD bound to RNAP, we identified amino acid substitutions that increase the affinity of CarD for RNAP. Using these substitutions, we show that increasing the affinity of CarD for RNAP increases the stability of the CarD protein in M. tuberculosis In addition, we show that increasing the affinity of CarD for RNAP increases the growth rate in M. tuberculosis without affecting 16S rRNA levels. We further show that increasing the affinity of CarD for RNAP reduces M. tuberculosis virulence in a mouse model of infection despite the improved growth rate in vitro Our findings suggest that the CarD-RNAP interaction protects CarD from proteolytic degradation in M. tuberculosis, establish that growth rate and rRNA levels can be uncoupled in M. tuberculosis and demonstrate that the strength of the CarD-RNAP interaction has been finely tuned to optimize virulence.
  
  
  
  Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major global health problem. In order to develop new strategies to battle this pathogen, we must gain a better understanding of the molecular processes involved in its survival and pathogenesis. We have previously identified CarD as an essential transcriptional regulator in mycobacteria. In this study, we detail the effects of increasing the affinity of CarD for RNAP on transcriptional regulation, CarD protein stability, and virulence. These studies expand our understanding of the global transcription regulator CarD, provide insight into how CarD activity is regulated, and broaden our understanding of prokaryotic transcription.
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Ashley L
authors.initials: AL
authors.lastname: Garner
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Jayan
authors.initials: J
authors.lastname: Rammohan
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Jeremy P
authors.initials: JP
authors.lastname: Huynh
authors.affiliation: Department of Genetics and Waksman Institute of Microbiology, Rutgers University, Piscataway, New Jersey, USA.
authors.forename: Lucas M
authors.initials: LM
authors.lastname: Onder
authors.affiliation: Laboratory of Molecular Biophysics, The Rockefeller University, New York, New York, USA.
authors.forename: James
authors.initials: J
authors.lastname: Chen
authors.affiliation: Laboratory of Molecular Biophysics, The Rockefeller University, New York, New York, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Bae
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Drake
authors.initials: D
authors.lastname: Jensen
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Weiss
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Ana Ruiz
authors.initials: AR
authors.lastname: Manzano
authors.affiliation: Laboratory of Molecular Biophysics, The Rockefeller University, New York, New York, USA.
authors.forename: Seth A
authors.initials: SA
authors.lastname: Darst
authors.affiliation: Laboratory of Molecular Biophysics, The Rockefeller University, New York, New York, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Campbell
authors.affiliation: Department of Genetics and Waksman Institute of Microbiology, Rutgers University, Piscataway, New Jersey, USA.
authors.forename: Bryce E
authors.initials: BE
authors.lastname: Nickels
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Galburt
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA stallings@wustl.edu.
authors.forename: Christina L
authors.initials: CL
authors.lastname: Stallings
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D011485:Protein Binding; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D012335:RNA, Ribosomal; D012321:DNA-Directed RNA Polymerases
keywords: Mycobacterium tuberculosis; RNA polymerase; protein stability; protein-protein interactions; rRNA; transcription initiation factor