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Structural basis of chloroplast transcription

ABSTRACT: Chloroplasts contain a dedicated genome, which encodes subunits of the photosynthesis machinery. Transcription of photosynthesis genes is predominantly carried out by a plastid-encoded RNA polymerase (PEP), a nearly 1 MDa complex composed of core subunits with homology to eubacterial RNA polymerases (RNAPs) and at least 12 additional chloroplast-specific PEP-associated proteins (PAPs). However, the architecture of this complex and the functions of the PAPs remain unknown. In this work, a 19-subunit PEP complex from Sinapis alba was purified by glycerol gradient centrifugation. All known subunits of the complex could be identified by LC-MS and the structure of the complex was determined by cryo-electron microscopy.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Sinapis Alba

TISSUE(S): Cotyledon, Leaf

SUBMITTER: Noah Ditz

LAB HEAD: Hans-Peter Braun

PROVIDER: PXD048976 | Pride | 2024-02-19

REPOSITORIES: Pride

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			Action	DRS
	MS_Results.7z	Other
	RpoC.raw	Raw
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Mass spectrometry-based proteomic characterization of plastid-encoded RNA polymerase purified from Sinapis alba.

Project description:RNA polymerases (RNAPs) transcribe DNA into RNA and are found in all living organisms with several degrees of complexity from single polypeptide chain to multimeric enzymes. In chloroplast of angiosperms, two RNAPs are involved in plastid gene transcription: the nuclear-encoded RNA polymerase (NEP) and the plastid-encoded RNA polymerase (PEP). The PEP is a prokaryotic-type multimeric RNAP found in different states depending on light stimuli and cell identity. One of these active states requires the assembly of nuclear-encoded proteins named PEP-Associated Proteins (PAPs) with the catalytic core, triggering the timely transcription of photosynthesis-associated plastid genes in cells acquiring their photosynthetic apparatus. A purification procedure was used to enrich native PEP from Sinapis alba chloroplasts. Mass spectrometry-based proteomic analysis of the obtained fraction identified the expected subunits, i.e. the core components and the PAPs as the most abundant proteins.

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Characterization vy crosslinking-mass spectrometry of plastid-encoded RNA polymerase purified from Sinapis alba.

Project description:RNA polymerases (RNAPs) transcribe DNA into RNA and are found in all living organisms with several degrees of complexity from single polypeptide chain to multimeric enzymes. In chloroplast of angiosperms, two RNAPs are involved in plastid gene transcription: the nuclear-encoded RNA polymerase (NEP) and the plastid-encoded RNA polymerase (PEP). The PEP is a prokaryotic-type multimeric RNAP found in different states depending on light stimuli and cell identity. One of these active states requires the assembly of nuclear-encoded proteins named PEP-Associated Proteins (PAPs) with the catalytic core, triggering the timely transcription of photosynthesis-associated plastid genes in cells acquiring their photosynthetic apparatus. A purification procedure was used to enrich native PEP from Sinapis alba chloroplasts. Crosslinking coupled to mass spectrometry provided initial structural information about the relative position of PEP subunits within the complex.

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Structure of the Plant Plastid-Encoded RNA Polymerase

Project description:In this study, the native Sinapis alba plastid-encoded RNA polymerase (PEP) complex was purified and cross-linking MS was used to help in its structure determination.

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pTAC3 and pTAC14 are required for binding of Plastid Encoded RNA Polymerase to DNA

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