One RNA polymerase serving two genomes

Abstract

The land plant Arabidopsis thaliana contains three closely related nuclear genes encoding phage-type RNA polymerases (RpoT;1, RpoT;2 and RpoT;3). The gene products of RpoT;1 and RpoT;3 have previously been shown to be imported into mitochondria and chloroplasts, respectively. Here we show that the transit peptide of RpoT;2 possesses dual targeting properties. Transient expression assays in tobacco protoplasts as well as stable transformation of Arabidopsis plants demonstrate efficient targeting of fusion peptides consisting of the N-terminus of RpoT;2 joined to green fluorescent protein to both organelles. Thus, RpoT;2 might be the first RNA polymerase shown to transcribe genes in two different genomes. RNA polymerase activity of recombinant RpoT;2 is uneffected by the inhibitor tagetin, qualifying the gene product of RpoT;2 as a phage-type polymerase.

Fig. 1. Transient expression of GFP fusions in wild-type tobacco protoplasts. (A) Expression of construct RpoT;2–GFP examined by CLSM. GFP fluorescence was detected in the green channel (left) and merged (right) with chlorophyll autofluorescence visible in the red channel. (B) Mitochondrial targeting control CoxIV–GFP (Akashi et al., 1998). (C) Plastid targeting control RecA–GFP (Akashi et al., 1998). The control images were taken by epifluorescence microscopy using GFP- (left) and FITC- (right) filter sets, respectively. Scale bars indicate 10 µm.

Fig. 2. Functional analysis of the two methionine residues in the N-terminus of RpoT;2. (A) Sequence comparison of of the three Arabidopisis RpoT gene products. Only the variable N-termini up to the motif RQVKXETE are shown. The alignment was performed using Multalin (Corpet, 1988); identical amino acid positions are highlighted. (B) Schematic presentation of the three different GFP fusions used in transient expression experiments (for details see Results). (C–E) Expression of RpoT;2–GFP (C), RpoT;2[M40I]–GFP (D), and RpoT;2[Δ39]–GFP (E) in protoplasts of the rpoB^– mutant of N. tabacum. Pictures were taken by CLSM in the green channel. Scale bars indicate 10 µm.

Fig. 3. Expression of the RpoT;2-mGFP5 in stably transformed Arabidopsis plants. (A) Fluorescence in root hair cells using the FITC-filter set. Under tissue culture conditions, some of the root plastids (arrowheads) accumulate photosynthetic pigments as evident by their yellowish color. (B–D) Counterstain of root tissue with MitoTracker-Red. GFP (B), and red MitoTracker (C) fluorescence were detected separately and merged (D) using Photoshop 5.0 software. Images were taken by epifluorescence microscopy using filter sets 488013 (B) and 488015 (C). Bars equal 10 µm.

Fig. 4. Purification and RNAP activity of recombinant RpoT;2. (A) SDS–PAGE of partially purified Trx-RpoT;2. Lanes: M, marker proteins, molecular masses in kDa; 1, uninduced E. coli lysate, soluble fraction; 2, induced cells, soluble fraction; 3, Ni-NTA-agarose purified fraction; 4, purified recombinant RNA polymerase after two-step dialysis purification. Arrow: 112 kDa recombinant Trx-RpoT;2. (B) RNA polymerase activity of Trx-RpoT;2 as incorporation of [³²P]UTP in vitro. Bars indicate relative RNA polymerase activity: black, no inhibitor; hatched, tagetin; gray, rifampicin; white, actinomycin D. Control reactions were performed using E. coli core RNA polymerase.