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Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics

Bacterial pathogens like Mycobacterium tuberculosis (Mtb) employ transcription factors to adapt their physiology to the diverse environments within their host. CarD is a conserved bacterial transcription factor that is essential for viability in Mtb. Unlike classical transcription factors that recog...

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Autores principales: Zhu, Dennis X., Stallings, Christina L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10232725/
https://www.ncbi.nlm.nih.gov/pubmed/37075846
http://dx.doi.org/10.1016/j.jbc.2023.104724
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author Zhu, Dennis X.
Stallings, Christina L.
author_facet Zhu, Dennis X.
Stallings, Christina L.
author_sort Zhu, Dennis X.
collection PubMed
description Bacterial pathogens like Mycobacterium tuberculosis (Mtb) employ transcription factors to adapt their physiology to the diverse environments within their host. CarD is a conserved bacterial transcription factor that is essential for viability in Mtb. Unlike classical transcription factors that recognize promoters by binding to specific DNA sequence motifs, CarD binds directly to the RNA polymerase to stabilize the open complex intermediate (RP(o)) during transcription initiation. We previously showed using RNA-sequencing that CarD is capable of both activating and repressing transcription in vivo. However, it is unknown how CarD achieves promoter-specific regulatory outcomes in Mtb despite binding indiscriminate of DNA sequence. We propose a model where CarD’s regulatory outcome depends on the promoter’s basal RP(o) stability and test this model using in vitro transcription from a panel of promoters with varying levels of RP(o) stability. We show that CarD directly activates full-length transcript production from the Mtb ribosomal RNA promoter rrnAP3 (AP3) and that the degree of transcription activation by CarD is negatively correlated with RP(o) stability. Using targeted mutations in the extended −10 and discriminator region of AP3, we show that CarD directly represses transcription from promoters that form relatively stable RP(o). DNA supercoiling also influenced RP(o) stability and affected the direction of CarD regulation, indicating that the outcome of CarD activity can be regulated by factors beyond promoter sequence. Our results provide experimental evidence for how RNA polymerase–binding transcription factors like CarD can exert specific regulatory outcomes based on the kinetic properties of a promoter.
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spelling pubmed-102327252023-06-02 Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics Zhu, Dennis X. Stallings, Christina L. J Biol Chem Research Article Bacterial pathogens like Mycobacterium tuberculosis (Mtb) employ transcription factors to adapt their physiology to the diverse environments within their host. CarD is a conserved bacterial transcription factor that is essential for viability in Mtb. Unlike classical transcription factors that recognize promoters by binding to specific DNA sequence motifs, CarD binds directly to the RNA polymerase to stabilize the open complex intermediate (RP(o)) during transcription initiation. We previously showed using RNA-sequencing that CarD is capable of both activating and repressing transcription in vivo. However, it is unknown how CarD achieves promoter-specific regulatory outcomes in Mtb despite binding indiscriminate of DNA sequence. We propose a model where CarD’s regulatory outcome depends on the promoter’s basal RP(o) stability and test this model using in vitro transcription from a panel of promoters with varying levels of RP(o) stability. We show that CarD directly activates full-length transcript production from the Mtb ribosomal RNA promoter rrnAP3 (AP3) and that the degree of transcription activation by CarD is negatively correlated with RP(o) stability. Using targeted mutations in the extended −10 and discriminator region of AP3, we show that CarD directly represses transcription from promoters that form relatively stable RP(o). DNA supercoiling also influenced RP(o) stability and affected the direction of CarD regulation, indicating that the outcome of CarD activity can be regulated by factors beyond promoter sequence. Our results provide experimental evidence for how RNA polymerase–binding transcription factors like CarD can exert specific regulatory outcomes based on the kinetic properties of a promoter. American Society for Biochemistry and Molecular Biology 2023-04-17 /pmc/articles/PMC10232725/ /pubmed/37075846 http://dx.doi.org/10.1016/j.jbc.2023.104724 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Zhu, Dennis X.
Stallings, Christina L.
Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics
title Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics
title_full Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics
title_fullStr Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics
title_full_unstemmed Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics
title_short Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics
title_sort transcription regulation by card in mycobacteria is guided by basal promoter kinetics
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10232725/
https://www.ncbi.nlm.nih.gov/pubmed/37075846
http://dx.doi.org/10.1016/j.jbc.2023.104724
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