The plastid genome of dicotyledonous plants is transcribed by three different RNA polymerases; an eubacterial-type enzyme, PEP; and two phage-type enzymes, RPOTp and RPOTmp. RPOTp plays an important role in chloroplast transcription, biogenesis, and mesophyll cell proliferation. RPOTmp fulfills a specific function in the transcription of the rrn operon in proplasts/amyloplasts during seed imbibition/germination and a more general function in chloroplasts during later developmental stages. In chloroplasts, RPOTmp is tightly associated with thylakoid membranes indicating that functional switching of RPOTmp is connected to thylakoid association. By using the yeast two-hybrid system, we have identified two proteins that interact with RPOTmp. The two proteins are very similar, both characterized by three N-terminal transmembrane domains and a C-terminal RING domain. We show that at least one of these proteins is an intrinsic thylakoid membrane protein that fixes RPOTmp on the stromal side of the thylakoid membrane, probably via the RING domain. A model is presented in which light by triggering the synthesis of the RING protein determines membrane association and functional switching of RPOTmp.chloroplast ͉ RING finger T ranscription of this small organellar genome is accomplished by three different RNA polymerases in a developmentally regulated manner (1, 2). One of the three RNA polymerases is of the eubacterial type, and its subunits are encoded on the plastid genome [plastid-encoded RNA polymerase (PEP)]. The two other RNA polymerases are nucleus-encoded [nucleus encoded plastid RNA polymerase (NEP)] and of the phage type (3, 4). Three different genes have been identified in Arabidopsis coding for NEP proteins that are localized in mitochondria (RPOTm, At1g68990), plastids (RPOTp, At2g24120), or in both organelles (RPOTmp, At5g15700; refs. 5 and 6). However, only two genes (RPOTp and RPOTm) have been detected in monocotyledons (4,7,8), thus raising the question of the function of RPOTmp in plastids of dicotyledon plants. Recent results have shown that RPOTp can be regarded as the principal NEP enzyme in chloroplasts playing an important role in chloroplast transcription, biogenesis, and mesophyll cell proliferation (9, 10). Results obtained with RPOTmp indicate partial replacement of RPOTp in RPOTp mutants (10, 11), an early function in lightinduced accumulation of several plastid mRNAs (12, 13), and specific transcription of the rrn operon of Arabidopsis from the PC promoter during seed imbibition and germination (10).Most of the plastid transcription units are preceded by NEP as well as PEP promoters and could be transcribed by the two types of RNA polymerase. However, with a few exceptions, NEP transcripts are barely detectable in mature chloroplasts, and most of the so-far-determined NEP promoters have been analyzed in PEP-deficient photosynthetically inactive plant material (14-17). These results led to an initial model of plastid transcription attributing special importance to NEP for the transcription of ho...

SummaryPlants 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, ®ve 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 ®fth 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-puri®es with both mitochondria and plastids. Its NH 2 -terminal 153 amino acids, translationally fused to green¯uorescent 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.

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