Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development

Sebastian Steiner,Yvonne Schröter,Thomas Pfannschmidt,Jeannette Pfalz

doi:10.1104/pp.111.184515

Sebastian Steiner, Yvonne Schröter + Show 2 more

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https://doi.org/10.1104/pp.111.184515

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Journal: Plant Physiology	Publication Date: Sep 23, 2011
Citations: 210	License type: CC BY 4.0

Affiliation: Friedrich Schiller University Jena

Abstract

The major RNA polymerase activity in mature chloroplasts is a multisubunit, Escherichia coli-like protein complex called PEP (for plastid-encoded RNA polymerase). Its subunit structure has been extensively investigated by biochemical means. Beside the "prokaryotic" subunits encoded by the plastome-located RNA polymerase genes, a number of additional nucleus-encoded subunits of eukaryotic origin have been identified in the PEP complex. These subunits appear to provide additional functions and regulation modes necessary to adapt transcription to the varying functional situations in chloroplasts. However, despite the enormous progress in genomic data and mass spectrometry techniques, it is still under debate which of these subunits belong to the core complex of PEP and which ones represent rather transient or peripheral components. Here, we present a catalog of true PEP subunits that is based on comparative analyses from biochemical purifications, protein mass spectrometry, and phenotypic analyses. We regard reproducibly identified protein subunits of the basic PEP complex as essential when the corresponding knockout mutants reveal an albino or pale-green phenotype. Our study provides a clearly defined subunit catalog of the basic PEP complex, generating the basis for a better understanding of chloroplast transcription regulation. In addition, the data support a model that links PEP complex assembly and chloroplast buildup during early seedling development in vascular plants.

Highlights

The major RNA polymerase activity in mature chloroplasts is a multisubunit, Escherichia coli-like protein complex called plastid-encoded RNA polymerase (PEP)
The PEP enzyme was further purified from such fractions by two-dimensional (2D) blue native (BN)-PAGE, as recently described (Schroter et al, 2010)
15 different protein bands were reproducibly identified in the PEP complex (Fig. 1), which were analyzed by mass spectrometry

Summary

Introduction

The major RNA polymerase activity in mature chloroplasts is a multisubunit, Escherichia coli-like protein complex called PEP (for plastid-encoded RNA polymerase). The PEP subunits, in contrast, are encoded by a set of plastomelocated genes (rpoA and the rpoB/C1/C2 operon) that exhibit approximately 26% to 50% sequence homology to corresponding genes from cyanobacteria, generating the so-called core enzyme (Igloi and Kossel, 1992) This core enzyme is supplemented by a number of nucleusencoded s-factors that provide the necessary promoter specificity to the complex (Link, 1996; Allison, 2000; Schweer et al, 2010). The first represents an insoluble RNA polymerase preparation called transcriptionally active chromosome (TAC), which can be precipitated by ultracentrifugation It represents a high-Mr DNA/RNA-protein complex containing approximately 40 to 60 proteins that is capable of in vitro transcription, resembling the nucleoids in this respect (Hallick et al, 1976; Reiss and Link, 1985; Little and Hallick, 1988; Krause and Krupinska, 2000; Pfalz et al, 2006). Many studies concentrated on these soluble preparations, since these allowed a precise molecular analysis of the promoter specificity and cis-element usage of the purified transcription complex (Bradley and Gatenby, 1985; Lerbs et al, 1985; Rajasekhar et al, 1991; Lakhani et al, 1992; Pfannschmidt and Link, 1997)

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