Dataset Information

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. IMPORTANCE: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.

SUBMITTER: Garner AL

PROVIDER: S-EPMC5287406 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Effects of Increasing the Affinity of CarD for RNA Polymerase on Mycobacterium tuberculosis Growth, rRNA Transcription, and Virulence.

Garner Ashley L AL Rammohan Jayan J Huynh Jeremy P JP Onder Lucas M LM Chen James J Bae Brian B Jensen Drake D Weiss Leslie A LA Manzano Ana Ruiz AR Darst Seth A SA Campbell Elizabeth A EA Nickels Bryce E BE Galburt Eric A EA Stallings Christina L CL

Journal of bacteriology 20170130 4

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, ...[more]

PMID: 27920294

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Project description:The first step in gene expression is the transcription of DNA sequences into RNA. Regulation at the level of transcription leads to changes in steady-state concentrations of RNA transcripts, affecting the flux of downstream functions and ultimately cellular phenotypes. Changes in transcript levels are routinely followed in cellular contexts via genome-wide sequencing techniques. However, in vitro mechanistic studies of transcription have lagged with respect to throughput. Here, we describe the use of a real-time, fluorescent-aptamer-based method to quantitate steady-state transcription rates of the Mycobacterium tuberculosis RNA polymerase. We present clear controls to show that the assay specifically reports on promoter-dependent, full-length RNA transcription rates that are in good agreement with the kinetics determined by gel-resolved, α- 32 P NTP incorporation experiments. We illustrate how the time-dependent changes in fluorescence can be used to measure regulatory effects of nucleotide concentrations and identity, RNAP and DNA concentrations, transcription factors, and antibiotics. Our data showcase the ability to easily perform hundreds of parallel steady-state measurements across varying conditions with high precision and reproducibility to facilitate the study of the molecular mechanisms of bacterial transcription.Significance statementRNA polymerase transcription mechanisms have largely been determined from in vitro kinetic and structural biology methods. In contrast to the limited throughput of these approaches, in vivo RNA sequencing provides genome-wide measurements but lacks the ability to dissect direct biochemical from indirect genetic mechanisms. Here, we present a method that bridges this gap, permitting high-throughput fluorescence-based measurements of in vitro steady-state transcription kinetics. We illustrate how an RNA-aptamer-based detection system can be used to generate quantitative information on direct mechanisms of transcriptional regulation and discuss the far-reaching implications for future applications.

Dataset Information

Effects of Increasing the Affinity of CarD for RNA Polymerase on Mycobacterium tuberculosis Growth, rRNA Transcription, and Virulence.

Publications

Effects of Increasing the Affinity of CarD for RNA Polymerase on Mycobacterium tuberculosis Growth, rRNA Transcription, and Virulence.

Similar Datasets

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