@article{682037ff79364b3cac4821c03ada209f,
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.",
keywords = "Mycobacterium tuberculosis, Protein stability, Proteinprotein interactions, RNA polymerase, RRNA, Transcription initiation factor",
author = "Garner, {Ashley L.} and Jayan Rammohan and Huynh, {Jeremy P.} and Onder, {Lucas M.} and James Chen and Brian Bae and Drake Jensen and Weiss, {Leslie A.} and Manzano, {Ana Ruiz} and Darst, {Seth A.} and Campbell, {Elizabeth A.} and Nickels, {Bryce E.} and Galburt, {Eric A.} and Stallings, {Christina L.}",
note = "Funding Information: We thank Tim Lohman for the use of his stopped-flow spectrophotometer, Tom Ellenberger for the use of the high-pressure and fast-performance liquid chromatography in his laboratory, and Yocheved Gensler for assistance with cloning. We are grateful to Katherine Mann and Jerome Prusa for their thoughtful review of the manuscript. We declare that we have no conflicts of interest with the contents of this article. C.L.S. and E.A.G. are supported by grant GM107544 from the National Institutes of Health. B.E.N. is supported by grant GM118059 from the National Institutes of Health. E.A.C. is supported by grant GM114450 from the National Institutes of Health. A.L.G. is supported by the NIGMS Cell and Molecular Biology Training grant GM007067 and the Stephen I. Morse Graduate Fellowship. J.R. is supported by a Sigma-Aldrich Predoctoral Fellowship. J.P.H. is supported by National Science Foundation Graduate Research Fellowship DGE-1143954. A.L.G., B.E.N., E.A.C., S.A.D., and C.L.S. conceived the project, designed experiments, analyzed results, and wrote the manuscript. A.L.G., J.R., J.P.H., L.A.W., L.M.O., B.B., J.C., D.J., and A.R.M. performed experiments and analyzed the results. E.A.G. analyzed results and edited the manuscript. Publisher Copyright: {\textcopyright} 2017 American Society for Microbiology.",
year = "2017",
doi = "10.1128/JB.00698-16",
language = "English",
volume = "199",
journal = "Journal of Bacteriology",
issn = "0021-9193",
number = "4",
}