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Cited by 17Photosynthetic Processes and MechanismsGenomics and Chromatin DynamicsBacterial Genetics and Biotechnology

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Three-Dimensional Envelope and Subunit Interactions of the Plastid-Encoded RNA Polymerase from Sinapis alba

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Ruedas, Rémi, Muthukumar, Soumiya Sankari, Kieffer-Jaquinod, Sylvie, Gillet, François-Xavier, Fenel, Daphna, Effantin, Grégory, Pfannschmidt, Thomas, Couté, Yohann, Blanvillain, Robert, Cobessi, David·2022·International Journal of Molecular Sciences
DOI 10.3390/ijms23179922PMC PMC9456514

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Abstract

RNA polymerases (RNAPs) are found in all living organisms. In the chloroplasts, the plastid-encoded RNA polymerase (PEP) is a prokaryotic-type multimeric RNAP involved in the selective transcription of the plastid genome. One of its active states requires the assembly of nuclear-encoded PEP-Associated Proteins (PAPs) on the catalytic core, producing a complex of more than 900 kDa, regarded as essential for chloroplast biogenesis. In this study, sequence alignments of the catalytic core subunits across various chloroplasts of the green lineage and prokaryotes combined with structural data show that variations are observed at the surface of the core, whereas internal amino acids associated with the catalytic activity are conserved. A purification procedure compatible with a structural analysis was used to enrich the native PEP from Sinapis alba chloroplasts. A mass spectrometry (MS)-based proteomic analysis revealed the core components, the PAPs and additional proteins, such as FLN2 and pTAC18. MS coupled with crosslinking (XL-MS) provided the initial structural information in the form of protein clusters, highlighting the relative position of some subunits with the surfaces of their interactions. Using negative stain electron microscopy, the PEP three-dimensional envelope was calculated. Particles classification shows that the protrusions are very well-conserved, offering a framework for the future positioning of all the PAPs. Overall, the results show that PEP-associated proteins are firmly and specifically associated with the catalytic core, giving to the plastid transcriptional complex a singular structure compared to other RNAPs.

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Metadata

Authors
Ruedas, R., Muthukumar, S. S., Kieffer-Jaquinod, S., Gillet, F.-X., Fenel, D., Effantin, G., Pfannschmidt, T., Couté, Y., Blanvillain, R., & Cobessi, D.
Affiliations
  • Centre National de la Recherche Scientifique (FR)
  • INSERM, CEA, University Grenoble Alpes, UMR BioSanté U1292, CNRS, CEA, FR2048, 38000 Grenoble, France
  • Lyon 1 Université (FR)
  • Leibniz University Hannover (DE)
Corresponding author
  • Blanvillain, Robert
  • Cobessi, David
ORCID
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2022-08-31
Volume
23
Issue
17
Pages
9922
ISSN
1422-0067 (ISSN-L)
DOI
10.3390/ijms23179922
PMC
PMC9456514
OpenAlex
W4294307745
Cited by
17
References
60
Citations by year
2026: 2 · 2025: 3 · 2024: 10 · 2023: 1 · 2022: 1
FWCI
1.22
Type
journal article
Language
en
Open access
Licence
Locations
  • International Journal of Molecular Sciences (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • the Agence National de la Recherche (ANR-17-CE11-0031-02, UMS 3518 CNRS-CEA-UGA-EMBL, ANR-10-INBS-0005-02, ANR-17-EURE-0003, ANR-10-INBS-08-01 grant)
  • the Grenoble Instruct-ERIC Centre (ANR-17-CE11-0031-02, UMS 3518 CNRS-CEA-UGA-EMBL, ANR-10-INBS-0005-02, ANR-17-EURE-0003, ANR-10-INBS-08-01 grant)
  • FRISBI (ANR-17-CE11-0031-02, UMS 3518 CNRS-CEA-UGA-EMBL, ANR-10-INBS-0005-02, ANR-17-EURE-0003, ANR-10-INBS-08-01 grant)
  • GRAL (ANR-17-CE11-0031-02, UMS 3518 CNRS-CEA-UGA-EMBL, ANR-10-INBS-0005-02, ANR-17-EURE-0003, ANR-10-INBS-08-01 grant)
  • the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) (ANR-17-CE11-0031-02, UMS 3518 CNRS-CEA-UGA-EMBL, ANR-10-INBS-0005-02, ANR-17-EURE-0003, ANR-10-INBS-08-01 grant)
  • ProFI (ANR-17-CE11-0031-02, UMS 3518 CNRS-CEA-UGA-EMBL, ANR-10-INBS-0005-02, ANR-17-EURE-0003, ANR-10-INBS-08-01 grant)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Photosynthetic Processes and Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
plastid-encoded RNA polymerase, chloroplast transcription, Sinapis alba, protein complex structure, subunit interactions, chloroplast biogenesis, cross-linking mass spectrometry, negative stain electron microscopy
MeSH terms
Chloroplasts, DNA-Directed RNA Polymerases, Plastids, Gene Expression Regulation, Plant, Arabidopsis Proteins, Sinapis, Proteomics

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