The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single-subunit enzyme or catalytic core of multimeric enzyme complexes?

Abstract

Highly purified RNA polymerase preparations from spinach chloroplasts contain seven major polypeptides of 150, 145, 110, 102, 80, 75, and 38 kDa. I find that RNA polymerase activity can be separated under defined conditions into three different fractions by heparin-Sepharose chromatography. Immunological analysis has shown that the rEst fraction contains RNA polymerase activity associated with all seven major polypeptides, and other studies have shown that some of these polypeptides (150, 145, 80, and 38 kDa) are associated with an RNA polymerase similar to the Escherichia coli enzyme. However, similar analyses of the remaining fractions show activity associated only with the 110-kDa polypeptide, suggesting the existence of a second kind of chloroplast RNA polymerase. Samples of this 110-kDa polypeptide purified by SDS/PAGE actively synthesize RNA in a reaction dependent on a supercoiled DNA template and the four ribonucleoside triphosphates. Hence, this polypeptide has all of the properties expected of a single-subunit RNA polymerase of the T7 bacteriophage type.Regulation of transcription in chloroplasts is still poorly understood. Originally, the existence of two different RNA polymerase activities was suggested by the finding that tRNA and mRNA genes in Euglena chloroplasts could be transcribed by a soluble enzymatic activity, whereas transcription of rRNA genes required a membrane-bound fraction (1). The activity of the soluble fraction was correlated with the expression of the rpo genes of the plastid genome, which are homologous to bacterial rpo genes that make up the bacterial RNA polymerase (2). However, neither ofthese fractions has been purified to homogeneity.The most highly purified chloroplast RNA polymerase (cp RNA polymerase) preparations obtained from maize, spinach, and pea contain 7-14 polypeptides (3)(4)(5)(6). In maize, some of these polypeptides (180, 120, 78, and 38 kDa) were shown to be products of the plastid rpo genes (5). In pea, some of these polypeptides (150, 54, and 51 kDa) have been confirmed as RNA polymerase subunits by photocrosslinking (7,8). In preparations of spinach cp RNA polymerase, all of the major polypeptides were identified as components of active cp RNA polymerases through use of antibody-linked polymerase assays (ALPAs; ref.3). However, these assays do not reveal whether these preparations contain only one active RNA polymerase or two.In previous studies it has been shown that spinach leaf homogenates contain polypeptides that are immunologically related to ,8p3', a, and o-subunits of Escherichia coli RNA polymerase. The latter two polypeptides are separated from the ,3/3' homologs during PEI-cellulose chromatography.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Specific initiation at the rbcL promoter requires both fractions, which supports the idea that this transcription is carried out by an RN...

elongated mesocotyl1, a Phytochrome-Deficient Mutant of Maize

“…The identification of nuclear-encoded plastidlocalized RNA polymerase (Greenberg et al, 1984;Lerbs-Mache, 1993;Young et al, 1998) and plastidtargeted sigma factors (Tiller et al, 1991;Lahiri et al, 1999) has revealed the importance of transcriptional control in the regulation in plastid gene expression. The accumulation of both nuclear-encoded plastidlocalized RNA polymerase and plastid-localized sigma factors is normally light regulated (Chang et al, 1999;Lahiri et al, 1999) and, therefore, may be disrupted in elm1 plants.…”

A tRNA gene transcription initiation site is similar to mRNA and rRNA promoters in plant mitochondria

“…In plastids rRNA genes are duplicated and evidence for an additional RNA-polymerase (and/or its respective cofactors) has been described, which may provide a differential tanscriptional activity (3)(4)(5). Only few of the plastid tRNA genes are located in the inverted repeat region, while most of the others appear to be cotranscribed with protein coding genes (6)(7)(8).…”

A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression

“…The transcriptional machinery plays an important role in the regulation of chloroplast development at different developmental stages (Mullet, 1993;Emanuel et al, 2004;Zoschke et al, 2007). Higher plant cells possess at least two types of RNA polymerases: a plastid-encoded bacterial-type RNA polymerase (PEP), which is a multisubunit eubacterium-type enzyme, and a nucleusencoded phage-type RNA polymerase (NEP) that resembles the single-subunit type shared with phage T3/T7 and mitochondrial enzymes (Hu and Bogorad, 1990;Igloi and Kö ssel, 1992;Lerbs-Mache, 1993;Pfannschmidt and Link, 1997;Liere and Maliga, 2001). The chloroplast-encoded photosynthetic genes are exclusively transcribed by PEP (class I genes).…”