Details
| Original language | English |
|---|---|
| Article number | 9922 |
| Journal | International Journal of Molecular Sciences |
| Volume | 23 |
| Issue number | 17 |
| Publication status | Published - Sept 2022 |
| Externally published | Yes |
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.
Keywords
- chloroplast biogenesis, PEP associated proteins, photomorphogenesis, photosynthesis, plastid-encoded RNA polymerase, Sinapis alba, transcription
ASJC Scopus subject areas
- Chemical Engineering(all)
- Catalysis
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
- Chemistry(all)
- Spectroscopy
- Computer Science(all)
- Computer Science Applications
- Chemistry(all)
- Physical and Theoretical Chemistry
- Chemistry(all)
- Organic Chemistry
- Chemistry(all)
- Inorganic Chemistry
Cite this
- Standard
- Harvard
- Apa
- Vancouver
- BibTeX
- RIS
In: International Journal of Molecular Sciences, Vol. 23, No. 17, 9922, 09.2022.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Three-Dimensional Envelope and Subunit Interactions of the Plastid-Encoded RNA Polymerase from Sinapis alba
AU - Ruedas, Rémi
AU - Muthukumar, Soumiya Sankari
AU - Kieffer-Jaquinod, Sylvie
AU - Gillet, François Xavier
AU - Fenel, Daphna
AU - Effantin, Grégory
AU - Pfannschmidt, Thomas
AU - Couté, Yohann
AU - Blanvillain, Robert
AU - Cobessi, David
N1 - Publisher Copyright: © 2022 by the authors.
PY - 2022/9
Y1 - 2022/9
N2 - 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.
AB - 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.
KW - chloroplast biogenesis
KW - PEP associated proteins
KW - photomorphogenesis
KW - photosynthesis
KW - plastid-encoded RNA polymerase
KW - Sinapis alba
KW - transcription
UR - http://www.scopus.com/inward/record.url?scp=85137609230&partnerID=8YFLogxK
U2 - 10.3390/ijms23179922
DO - 10.3390/ijms23179922
M3 - Article
C2 - 36077319
AN - SCOPUS:85137609230
VL - 23
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
SN - 1661-6596
IS - 17
M1 - 9922
ER -