Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme.

We affinity-purified the tobacco plastid-encoded plastid RNA polymerase (PEP) complex by the alpha subunit containing a C-terminal 12 x histidine tag using heparin and Ni(2+) chromatography. The composition of the complex was determined by mass spectrometry after separating the proteins of the >900 kDa complex in blue native and SDS polyacrylamide gels. The purified PEP contained the core alpha, beta, beta', beta" subunits and five major associated proteins of unknown function, but lacked sigma factors required for promoter recognition. The holoenzyme efficiently recognized a plastid psbA promoter when it was reconstituted from the purified PEP and recombinant plastid sigma factors. Reconstitution of a plastid holoenzyme with individual sigma factors will facilitate identification of sigma factor-specific promoter elements.

A nucleus-encoded maize protein with sigma factor activity accumulates in mitochondria and chloroplasts.

Plants contain nuclear gene families that encode proteins related to the principal sigma factors of eubacteria. As sigma factors function in transcription, the plant proteins have been presumed or demonstrated to associate with the eubacteria-like RNA polymerase of chloroplasts. In maize, five sig cDNA sequences have been reported, and four of the products are present in plastids as predicted. However, in vitro chloroplast import assays and computer algorithms gave ambiguous results with the fifth protein, ZmSig2B. Unlike the other maize sigma factors, ZmSig2B is expressed throughout developing seedling leaves, as well as in roots and etiolated tissues. To determine the subcellular location of ZmSig2B, we have now used immunoblot assays to show that it co-purifies with both mitochondria and plastids. Its NH2-terminal 153 amino acids, translationally fused to green fluorescent protein (GFP), targeted GFP to chloroplasts and mitochondria in bombarded maize leaves. A putative role for ZmSig2B in mitochondrial transcription is supported by its presence in a maize mitochondrial transcription extract. ZmSig2B also exhibits the expected properties of a chloroplast sigma factor: recombinant ZmSig2B binds to a chloroplast promoter and initiates transcription in vitro when combined with Escherichia coli core RNA polymerase. Therefore ZmSig2B is an unusual nucleus-encoded sigma factor that appears to function in both chloroplasts and mitochondria.