Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis

Herrera-Asmat, Omar; Lubkowska, Lucyna; Kashlev, Mikhail; Bustamante, Carlos; Guerra, Daniel G.; Kireeva, Maria L.

doi:10.1016/j.pep.2017.03.013

Cited by 7 publications

(6 citation statements)

References 38 publications

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“…M. tuberculosis RNAP was prepared from cultures of E. coli strain BL21(DE3) (Invitrogen, Inc.) co-transformed with plasmids of pACYC Duet- rpoA-rpoD , pCDF- rpoZ and pET Duet- rpoB-rpoC , and purified as described with some modifications (39). Single colonies of the resulting transformants were used to inoculate 100 mL LB broth containing 35 μg/mL chloramphenicol, 100 μg/mL ampicillin, 50 μg/mL streptomycin, and cultures were incubated for 16 h at 37 °C with shaking.…”

Section: Methodsmentioning

confidence: 99%

Structural insights into transcription activation mechanism of the global regulator GlnR from actinobacteria

Lin

Shi

Feng

et al. 2023

Preprint

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GlnR, an OmpR/PhoB subfamily protein, is an orphan response regulator that globally coordinates the expression of genes responsible for nitrogen, carbon and phosphate metabolism in actinobacteria. Although much efforts at biochemical and genetic analyses have been made on the mechanism of GlnR-dependent transcription activation, it still remains unclear owing to lacking the structure of GlnR-dependent transcription activation complex (GlnR-TAC). Here, we report a crystal structure of a binary complex including a C terminal DNA binding domain of GlnR (GlnR_DBD) and its regulatory cis-element DNA, and a cryo-EM structure of GlnR-TAC comprising of Mycobacterium tuberculosis RNA polymerase, GlnR, and a promoter containing four well-characterized conserved GlnR binding sites. These structures show four GlnR protomers coordinately engage promoter DNA in a head-to-tail manner, with two N-terminal receiver domains of GlnR (GlnR-RECs) jointly act as a bridge to connect RNAP αNTD with the upstream GlnR_DBD. GlnR-TAC is stabilized by complex protein-protein interactions between GlnR and the conserved beta flap, σAR4, alphaCTD, alphaNTD domains of RNAP. These are in good agreement with our mutational and kinetic single-molecule fluorescence assays. Altogether, our results reveal a general transcription activation mechanism for the global regulator GlnR and other OmpR/PhoB subfamily proteins, and present a unique mode of bacterial transcription regulation.

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Section: Methodsmentioning

confidence: 99%

Structural insights into transcription activation mechanism of the global regulator GlnR from actinobacteria

Lin

Shi

Feng

et al. 2023

Preprint

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“…Although its homologs (RpoK and RPB6) are essential in archaea and eukaryotes (28), has been shown to be nonessential in several bacteria (29)(30)(31)(32). In M. tuberculosis, the roles of have not been clearly investigated (33)(34)(35). In this study, we showed that the subunit is essential for the assembly of M. tuberculosis RNAP core and have characterized a loop region in M. tuberculosis which is essential for its roles.…”

Section: Discussionmentioning

confidence: 74%

Association of ω with the C-Terminal Region of the β′ Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis

Mao

Zhu

et al. 2018

J Bacteriol

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The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Unlike the well-studied RNAP, the RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of ω with ω subunits from or cannot restore the assembly of RNAP. Furthermore, by replacing different regions in ω with the corresponding regions from ω, we found a nonconserved loop region in ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the β' subunit (β'CTD) in RNAP but not in or RNAP is close to the ω loop region. Deletion of this β'CTD in RNAP destabilized the binding of ω on RNAP and compromised core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in Sequence alignment of the ω loop and the β'CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of ω and highlighted the importance of the ω loop region in RNAP assembly. DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (αββ'ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. In and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen, and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.

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“…We observed that deleting the N-terminal fourteen residues involved in domain-swapping in CarD results in loss of self-association to form SDS-resistant higher order oligomers and hampers its ability to form amyloids in solution. Though in our solution studies we did not observe monomeric form of CarD under tested concentrations, Asmat et al ., 52 have reported a small population of monomeric CarD in gel filtration experiments. The crystal structure of 1:1 stoichiometric complex of Mtb RNAP β1β2/CarD complex suggests that CarD exists as monomer as well in solution 18 .…”

Section: Discussionmentioning

confidence: 99%

Mycobacterium tuberculosis CarD, an essential global transcriptional regulator forms amyloid-like fibrils

Kaur

Kaundal

Kapoor

et al. 2018

Sci Rep

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CarD is an essential global transcription regulator from Mycobacterium tuberculosis (Mtb) that binds RNA polymerase and activates transcription by stabilizing the transcription initiation complex. Available crystal structures have captured two distinct, monomeric and domain-swapped homodimeric, oligomeric states of CarD. However, the actual oligomeric state of CarD in solution and its biological relevance has remained unclear. Here, we confirm the presence of the homodimeric state of CarD in solution by using synchrotron-based small-angle X-ray scattering. Furthermore, by using biochemical and biophysical experiments, in addition to mass-spectrometry, transmission electron microscopy, and confocal imaging, we show that CarD is the first soluble cytosolic protein in Mtb which displays the tendency to form amyloid-like fibrils both in vitro as well as in vivo. We demonstrate that the deletion of the fourteen N-terminal residues involved in domain-swapping hampers amyloid formation, thus, suggesting that domain-swapping is crucial in amyloidogenesis. The discovery of the amyloidogenic property of an essential cytosolic global transcription regulator, CarD, in a pathogenic bacteria will further open up new frontiers in research.

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Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis

Cited by 7 publications

References 38 publications

Structural insights into transcription activation mechanism of the global regulator GlnR from actinobacteria

Structural insights into transcription activation mechanism of the global regulator GlnR from actinobacteria

Association of ω with the C-Terminal Region of the β′ Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis

Mycobacterium tuberculosis CarD, an essential global transcriptional regulator forms amyloid-like fibrils

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