Evolution of the mitochondrion was in nearly all organisms accompanied by the loss of genes encoding the bacterial-type RNA polymerase and the acquisition of a different transcription apparatus, the protein components of which are encoded in the nucleus and imported into the organelle (reviewed in (Gray and Lang, 1998;Hess and Börner, 1999;Tracy and Stern, 1995). Excepting the brown alga Pylaiella littoralis, none of the mitochondrial genomes of photosynthetic eukaryotes sequenced to date harbours sequence motifs of bacterial-type σ⁷⁰-dependent promoters. Instead, promoters of diverse architecture have been identified (see I.3.1). Mitochondrial RNA polymerases accordingly differ from enzymes of the bacterial type.

In Saccharomyces cerevisiae, the nuclear RPO41 gene encodes a phage-type RNA polymerase operating as catalytic subunit of the mitochondrial transcription machinery (Greenleaf, et al., 1986;Masters, et al., 1987); see Figure 4). A phage-type enzyme was moreover shown to function as core RNA polymerase in mitochondrial transcription in humans (Falkenberg, et al., 2002;Tiranti, et al., 1997). DNA sequences homologous to mitochondrial phage-type and bacteriophage T3/T7 RNA polymerases have been amplified from a phylogenetically broad range of multicellular and unicellular eukaryotes, suggesting that a phage-type enzyme was recruited to function in mitochondrial transcription at an early stage in the evolution of the mitochondrion (Cermakian, et al., 1996).

Genes encoding T3/T7 phage-like RNA polymerases, which are commonly designated RpoT genes, have been identified in the nuclear genomes of various angiosperms such as Chenopodium album (Weihe, et al., 1997), Arabidopsis (Hedtke, et al., 1997;Hedtke, et al., 2000), maize (Chang, et al., 1999;Young, et al., 1998), wheat (Ikeda and Gray, 1999), Nicotiana tabacum(Hedtke, et al., 2002), Nicotiana sylvestris(Kobayashi, et al., 2002;Kobayashi, et al., 2001), barley (Emanuel, et al., 2004), and in the moss Physcomitrella patens(Kabeya, et al., 2002;Richter, et al., 2002). Homologous sequences were moreover detected in the gymnosperm Pinus taeda (U. Richter, HU Berlin, personal communication) and in green algae (A. Weihe, HU Berlin, personal communication).

According to in vitro and in vivo import studies, a small family of three RpoT genes in Arabidopsis encodes a mitochondrial RNA polymerase (RpoTm), a plastidial enzyme (RpoTp) as well as a polypeptide imported into both mitochondria and plastids (RpoTmp; see Figure 4) (Hedtke, et al., 1997;Hedtke, et al., 2000;Hedtke, et al., 1999). Comparable subcellular targeting of RpoT gene products is observed in Nicotiana species (Hedtke, et al., 2002;Kobayashi, et al., 2001). In contrast, monocots have so far been determined to harbour no more than two RpoT genes, of which one codes for a mitochondrial RNA polymerase and the other for a plastidial enzyme (Figure 4; (Chang, et al., 1999;Ikeda and Gray, 1999). Dual targeting of RpoT gene products to both mitochondria and plastids being the result of two different translational starts has been reported for the two phage-type RNA polymerases genes RpoT1 and RpoT2 of Physcomitrella (Figure 4; (Richter, et al., 2002). For both RpoT1 and RpoT2, translation initiation at the first of two in-frame AUG start codons was found to yield a polypeptide that is targeted to plastids, whereas initiation at the downstream AUG gave rise to a mitochondrial protein (Richter, et al., 2002). In vivo translation initiation at the first AUG and a plastidial localization of both RpoT1 and RpoT2 in Physcomitrella and of RpoTmp in Arabidopsis have recently been questioned by (Kabeya and Sato, 2005) and await further experimental proof.

Figure 4: Nuclear genes encoding organellar phage-type RNA polymerases.

Genes in the nucleus (N, grey) of eukaryotic organisms code for T7 phage-like RNA polymerases which, following their synthesis in the cytoplasm, are imported into mitochondria (M, yellow) and plastids (P, green).

The small RpoT gene families found in Physcomitrella and in higher plants appear to be the result of independent gene duplication events dating after the separation of bryophytes from the vascular plant lineage (Richter, et al., 2002). Phylogenetic analyses have shown all higher plant RpoTp enzymes to form a sister clade to the group of RpoTm and RpoTmp enzymes (Richter, et al., 2002). Interestingly, preliminary phylogenetic analyses are indicative of a closer relationship of monocot RpoTm to dicot RpoTmp than to dicot RpoTm (U. Richter, HU Berlin, personal communication).

A DNA polymerase or reverse transcriptase has been proposed as predecessor of a single-subunit RNA polymerase that was the common ancestor of phage-encoded and nucleus-encoded phage-type RNA polymerases (Cermakian, et al., 1997). The ancestral single-subunit RNA polymerase gene has moreover been suggested to have originated at essentially the same time as the mitochondriate eukaryotic cell (Cermakian, et al., 1997). The discovery of cryptic prophages related to T3/T7 bacteriophages in several genomes of proteobacteria has inspired a modified scenario: A prophage that encoded among other phage proteins a T3/T7-like RNA polymerase existed in the bacterial endosymbiont being the predecessor of the mitochondrion (Filee and Forterre, 2005). Transfer of the prophage genes to the nucleus may have resulted in the reactivation of these formerly silent genes (Filee and Forterre, 2005).

Distinct functions of RpoTm and RpoTmp in mitochondria and of RpoTp and RpoTmp in plastids of dicots are yet to be assigned. The RpoTm and RpoTmp genes in Arabidopsis have been reported to display overlapping expression patterns in different tissues and at different developmental stages (Emanuel, et al., 2005). Emanuel et al. (2005) therefore suggested RpoTm and RpoTmp to recognize different types of mitochondrial promoters. A contrasting picture of RpoTm and RpoTmp functions has been stimulated by studies of a transgenic Arabidopsis line carrying a T-DNA insertion in the RpoTmp gene (Baba, et al., 2004). Transgenic plants displayed no apparent alterations compared to wild-type individuals in mitochondrial transcript accumulation. Based predominantly on the observation that in the mutant, the induction of several plastid genes in dark-grown seedlings upon illumination was delayed, Baba et al. (2004) proposed RpoTmp to be the key RNA polymerase transcribing organellar genes during early seedling development and favoured a role of both RpoTm and RpoTp at a later developmental stage.

Only indirect evidence has been provided that the mitochondrial phage-type RNA polymerases encoded by RpoT genes in higher plants have a role in transcription of mitochondrial genes. No sequences that might encode potential mitochondrial RNA polymerases of known enzyme structure have been traced in the fully sequenced Arabidopsis genome besides the previously characterized genes RpoTm and RpoTmp(Hedtke, et al., 1997;Hedtke, et al., 2000;Initiative, 2000). The conservation of functionally critical amino acid positions of the T7 enzyme (McAllister and Raskin, 1993;Sousa, et al., 1993) in RpoTm and RpoTmp as well as in other plant RpoT enzymes argues for their transcriptional function (Hess and Börner, 1999); see I.3.2.3 and Figure 5). Moreover, recombinant RpoTm and RpoTmp were shown to non-specifically transcribe DNA in vitro(Hedtke, et al., 2000;Kühn, 2001).

The mitochondrial RNA polymerase functions as primase for mtDNA replication in humans and presumably also in yeast (reviewed in (Shadel and Clayton, 1997;Tracy and Stern, 1995). A similar role of phage-type RNA polymerases and association of origins of replication with mitochondrial transcription start sites in plants remain to be demonstrated.

The RNA polymerase of the T7 bacteriophage is a 99-kDa single-polypeptide-chain enzyme that is able to recognize specific promoter sequences, correctly initiate transcription and catalyze transcript elongation (reviewed in (Steitz, 2004). Although the phage-type RNA polymerases of eukaryotic organisms most probably require auxiliary proteins to initiate transcription at organellar promoters (see I.3.3), the thoroughly studied T7 enzyme commonly serves as a model for both bacteriophage and eukaryotic phage-type RNA polymerases.

Comparisons between amino acid sequences of phage- and nucleus-encoded enzymes have identified conserved domains comprising identical or conservatively substituted positions (Chang, et al., 1999;Hedtke, 1998). Catalytically relevant structures of the T7 RNA polymerase are formed through amino acids of the C-terminal half of the polypeptide (McAllister and Raskin, 1993;Sousa, et al., 1993). Crystal structures of the T7 RNA polymerase have revealed this portion of the protein to fold into the “fingers”, “palm” and “thumb” subdomains (Jeruzalmi and Steitz, 1998;Sousa, et al., 1993); see Figure 5) that are typical to members of a superfamily of nucleic acid polymerases also including certain DNA polymerases and reverse transcriptases (reviewed in (Sousa, 1996). The similarity of plant organellar RNA polymerases to the T7 enzyme is most apparent for sequence regions corresponding to palm and fingers, which include all essential residues that participate in catalysing RNA synthesis (Figure 5; (Hess and Börner, 1999), and references therein). In contrast, elements contributing to promoter recognition by phage RNA polymerases are poorly conserved in plant phage-type transcriptases, possibly reflecting the divergence in architecture between promoters recognized by the two groups of enzymes, as well as different compositions of initiating RNA polymerase complexes (Chang, et al., 1999;Ikeda and Gray, 1999;Jeruzalmi and Steitz, 1998); see I.3.3). Based on structural modelling, Yeast Rpo41 has been suggested to possess two regions that correspond in structure, yet not in sequence, to the “specificity loop” and intercalating β-hairpin involved in promoter recognition and DNA
melting by T7 RNA polymerase (Matsunaga and Jaehning, 2004). In the T7 RNA polymerase-promoter complex, an antiparallel β-hairpin referred to as specificity loop specifically interacts via hydrogen-bonding with bases of the promoter sequence (Cheetham, et al., 1999;Rong, et al., 1998). The intercalating β-hairpin, which is part of the N-terminal domain of the T7 enzyme, facilitates melting of the promoter duplex (Cheetham, et al., 1999). Rpo41 was recently shown to accurately initiate promoter-specific transcription in vitro from supercoiled and pre-melted DNA templates in the absence of the obligatory yeast mitochondrial transcription factor sc-mtTFB, indicating that promoter specificity determinants indeed reside in the Rpo41 polypeptide rather than in sc-mtTFB (Matsunaga and Jaehning, 2004), see I.3.3.1).

Differences in size between nucleus-encoded phage-type RNA polymerases are primarily accounted for by varying N-terminal extensions. Including transit peptides, T7-like transcriptases of plants are proteins of around 110 kDa, whereas the yeast and human mitochondrial RNA polymerases are 145 and 130 kDa in size (Hess and Börner, 1999), and references therein). The N-terminal extension of yeast Rpo41 and a C-terminal insertion found only in the yeast enzyme are required for stable mtDNA maintenance and represent independent functional domains that may have been acquired through gene fusion events (Lisowsky, et al., 2002;Wang and Shadel, 1999). In heterologous complementation experiments, Arabidopsis RpoTm and various Rpo41/RpoTm chimeras were unable to functionally substitute for Rpo41 in vivo(Lisowsky, et al., 2002).

A number of separate crystal structures have depicted T7 RNA polymerase at different stages from promoter binding to elongation (Cheetham, et al., 1999;Cheetham and Steitz, 1999;Tahirov, et al., 2002;Yin and Steitz, 2002;Yin and Steitz, 2004). Following promoter rcognition, duplex DNA opening and repeated abortive initiation attempts, a major conformational change in the N-terminal domain removes the promoter-binding site and creates a tunnel for the transcript to pass through during the elongation phase in which the enzyme completes the RNA product processively without dissociation until termination (Steitz, 2004). Transcription termination signals recognized by the T7 RNA polymerase have been characterized (He, et al., 1998;Lyakhov, et al., 1998;Macdonald, et al., 1994), whereas no such sequences have been described in organelles. Mitochondrial transcripts in plants are instead considered to be terminated through the action of nucleases that define RNA 3’ ends (Dombrowski, et al., 1997;Perrin, et al., 2004).

Figure 5: Conserved sequence regions of the T7 phage and eukaryotic phage-type RNA polymerases.

(A) Surface representation of the T7 RNA polymerase-promoter complex structure specifying the N-terminal domain (yellow) and the RNA polymerase subdomains “thumb” (green), “palm” (red) and “fingers” (blue; from (Cheetham, et al., 1999). (B) Amino acid sequence organization of T7 RNA polymerase domains (colours as in Figure 5A) and homologous amino acid sequence regions of organellar phage-type RNA polymerases, based on a sequence comparison derived in (Hedtke, 1998). Arabidopsis RpoTm is shown as one member of the group of mitochondrial and plastidial enzymes of land plants which do not greatly differ in their sequence organization and size of the N-terminus. Black squares mark the positions of the motifs T/DxxGR (III), A, B and C (according to (Delarue, et al., 1990) that are important for RNA polymerase function and conserved in all enzymes, and of a T7-specific β-hairpin (I) and a structure involved in recognition of A/T-rich promoter regions (II). Open triangles denote the invariant residues Asp537 and Asp812 acting as ligands to two catalytic Mg²⁺ ions at the RNA polymerase active site (Woody, et al., 1996). The region corresponding to the specificity loop of the fingers subdomain is depicted in light blue; a yeast-specific C-terminal insertion is shown in grey.

In addition to the core RNA polymerase Rpo41, transcription initiation at mitochondrial promoters in S. cerevisiae requires a single accessory protein of 43 kDa first described as Mtf1 (Figure 6; (Lisowsky and Michaelis, 1988;Schinkel, et al., 1987) and also referred to as sc-mtTFB (Shadel and Clayton, 1993). While neither Rpo41 nor sc-mtTFB was able on its own to specifically interact with promoter sequences in gel mobility shift assays (Schinkel, et al., 1988), the non-specifically transcribing core was found to recognize mitochondrial promoters on a linear DNA template when complemented with sc-mtTFB in in vitro transcription experiments (Schinkel, et al., 1987). The latter was therefore considered the specificity factor of Rpo41 (Schinkel, et al., 1987). Functional studies of mutant variants of
sc-mtTFB motivated the alternative view that specificity determinants of promoter recognition reside in the core enzyme, and that sc-mtTFB may play a role in unwinding DNA during transcription initiation (Shadel and Clayton, 1995). Intrinsic promoter specificity of the core RNA polymerase was later confirmed not only for yeast Rpo41 (Matsunaga and Jaehning, 2004) but in part also for the human and mouse core enzymes (Gaspari, et al., 2004). On supercoiled or premelted DNA templates, Rpo41 alone was able to accurately initiate transcription in vitro at mitochondrial promoters (Matsunaga and Jaehning, 2004). While sc-mtTFB was obligatory for specific transcription of linear templates, addition of
sc-mtTFB stimulated specific transcription from supercoiled DNA but inhibited escape into
productive elongation from a pre-melted promoter (Matsunaga and Jaehning, 2004). The authors concluded that sc-mtTFB facilitated DNA melting, but not promoter recognition, possibly by inducing and stabilizing structural changes in Rpo41 that enable the enzyme to open the double helix and form an open promoter complex.

Figure 6: Components of the transcription machineries in yeast and human mitochondria.

Yeast transcription initiation model after (Shadel and Clayton, 1993); transcription initiation in human mitochondria as proposed by McCulloch and Shadel (2003). See text for details on mtTFB and mtTFA functions and cofactor interactions with the mitochondrial phage-type RNA polymerases Rpo41 and h-mtRNApol. Cofactor designations adhere to the nomenclature suggested by Shadel and Clayton (1993). mtTFA-induced DNA bending is indicated; bent arrows mark transcriptional starts.

Analyses of the RNA polymerase composition before, during and shortly after transcription initiation indicated that the Rpo41 core and sc-mtTFB form a holoenzyme in solution prior to DNA binding and promoter recognition (Mangus, et al., 1994). Upon binding to the promoter, the RNA polymerase holoenzyme induces a bend in the DNA, which has been shown to enhance promoter activity in vitro(Schinkel, et al., 1988). Following transcription initiation, the factor dissociates from the catalytic subunit and is available for subsequent initiation events (Mangus, et al., 1994). The observation that sc-mtTFB behaved like bacterial sigma factors during transcription initiation stimulated a series of experiments investigating functional and structural similarities between sigma factors and sc-mtTFB (Cliften, et al., 2000;Cliften, et al., 1997;Jang and Jaehning, 1991). However, resolution of the three-dimensional structure of sc-mtTFB later revealed this mitochondrial transcription factor to structurally resemble S-adenosyl-l-methionine (SAM)-dependent rRNA dimethylases such as the Bacillus subtilis rRNA methyltransferase ErmC’ (Schubot, et al., 2001).

Sequence similarity, albeit limited, of mitochondrial transcription factors to rRNA dimethylases became apparent following the identification of two sequences encoding mitochondrial sc-mtTFB-like factors in humans (Falkenberg, et al., 2002;McCulloch, et al., 2002). Homologues to human mtTFB1 and mtTFB2 (referred to as h-mtTFB1 and h-mtTFB2) where subsequently detected in mouse (Rantanen, et al., 2003) and Drosophila(Matsushima, et al., 2005;Matsushima, et al., 2004). Both h-mtTFB1 and h-mtTFB2 were found to form a stable heterodimer with human mitochondrial RNA polymerase and to induce transcription initiation at the human mtDNA promoters LSP and HSP in vitro in the presence of h-mtTFA (Figure 6; (Falkenberg, et al., 2002), an additional essential cofactor of human mitochondrial transcription (see I.3.3.2).

Despite poor overall sequence similarity of sc-mtTFB to ErmC’, there is extensive structural agreement between the two proteins around the SAM-binding site, and amino acid residues involved in SAM binding are conserved in sc-mtTFB (Schubot, et al., 2001). Therefore, Schubot et al. (2001) suggested that sc-mtTFB could bind SAM and methylate the nascent RNA chain in vivo. Alternatively, an RNA-modifying enzyme may have evolved to function solely as transcription factor. While no methyltransferase activity of sc-mtTFB or
h-mtTFB2 has been demonstrated so far, h-mtTFB1 has been reported to bind SAM and to substitute for the Escherichia coli rRNA dimethylase KsgA in methylating the 16S rRNA at a conserved stem-loop (McCulloch, et al., 2002;Seidel-Rogol, et al., 2003). The homologous 12S rRNA in human mitochondria was moreover shown to be similarly modified at this site (Seidel-Rogol, et al., 2003). Analyses of transcriptionally competent h-mtTFB1 variants carrying point mutations in conserved methyltransferase motifs indicated that the ability of the protein to function as transcription factor in vitro is independent of SAM binding and methyltransferase activity (McCulloch and Shadel, 2003). The authors therefore concluded
h-mtTFB1 to be a bifunctional protein with two separable activities.

In order to accurately and efficiently initiate transcription at the LSP and HSP promoters of the human mtDNA, the heterodimer composed of h-mtTFB and core RNA polymerase depends on the presence of the cofactor h-mtTFA (Falkenberg, et al., 2002;Fisher and Clayton, 1985;Fisher and Clayton, 1988). h-mtTFA is a protein of 25 kDa that comprises two HMG boxes in tandem (Parisi and Clayton, 1991). HMG boxes have been characterized as functional domains of DNA-binding proteins and interact with the minor grove of the double helix primarily at sites of unusual DNA conformation, where they induce dramatic bending and structural deformation of the DNA (Antoshechkin, et al., 1997;Giese, et al., 1997;Grosschedl, et al., 1994;Love, et al., 1995). h-mtTFA specifically binds to sequences upstream of the transcription initiation sites, which have been characterized as distal promoter elements (Fisher, et al., 1987;Gaspari, et al., 2004). Specific promoter binding by h-mtTFA as well as exact spacing between distal promoter elements and transcription start sites were shown to be crucial for transcription activation (Dairaghi, et al., 1995).

A h-mtTFA homologue is abundantly found in mitochondria of S. cerevisiae(Diffley and Stillman, 1991). sc-mtTFA is dispensable for transcription initiation at promoters of the yeast mtDNA (Xu and Clayton, 1992) and instead appears to play a major role in structural organization and stable maintenance of the mtDNA (Diffley and Stillman, 1991). Like
h-mtTFA, sc-mtTFA is able to specifically associate with regulatory sequences of the mtDNA and also non-specifically bind DNA, and mediates condensation and unwinding as well as bending of the double helix (Diffley and Stillman, 1992;Fisher, et al., 1992). Transcription initiation by the RNA polymerase holoenzyme is slightly stimulated by sc-mtTFA (Parisi, et al., 1993). Presumably, binding of sc-mtTFA to the mtDNA leads to a favourable exposition of cis-regulatory elements to the transcription apparatus (Diffley and Stillman, 1992).

The capacity of h-mtTFA to act as efficient and promoter sequence-specific transcriptional activator has been attributed to a C-terminal extension of the factor and a linker peptide between the two HMG boxes, which are both lacking in sc-mtTFA (Dairaghi, et al., 1995). Transfer of the C-terminal tail of h-mtTFA onto the yeast factor was shown to turn sc-mtTFA into a transcription factor activating the human mtDNA promoter LSP (Dairaghi, et al., 1995). Thus, h-mtTFA function appears to have evolved through the acquisition of novel structural domains. The C-terminal tail of h-mtTFA has been proposed to interact with h-mtTFB, thereby positioning the heterodimer composed of h-mtTFB and core RNA polymerase at the transcription initiation site demarcated by a specific h-mtTFA/promoter complex (Figure 6, (McCulloch and Shadel, 2003). Corroborating this model, promoter selectivity of the mouse and human transcription machineries has been dissected into binding of mtTFA to distal promoter elements and specificity of the core enzyme for nucleotides proximal to the transcription initiation site (Gaspari, et al., 2004).

To date, no mtTFA or mtTFB homologues have been isolated from plant mitochondria, and the function of such proteins in plant organelles is unclear. Attempts to detect HMG box proteins in maize mitochondrial extracts using a sc-mtTFA antibody did not identify mtTFA candidates (Andrea T. Descheneau, University of Missouri, Columbia, USA; personal communication). Moreover, application of a protocol that had been successfully employed for the preparation of mtTFA from yeast and human mitochondria failed to purify homologous proteins from pea mitochondria (Däschner, et al., 2001;Hatzack, et al., 1998).

A biochemical approach directed at isolating transcription factors from wheat mitochondria lead to the identification of p63, a 63-kDa protein described to specifically bind to the yeast cox2 promoter (Ikeda and Gray, 1999). Addition of recombinant p63 to a transcriptionally active extract prepared from wheat mitochondria, which per se accurately initiated transcription at the cox2 promoter, appeared to stimulate transcription from this promoter in vitro(Ikeda and Gray, 1999). The authors therefore suggested p63 to play a role in mitochondrial transcription and moreover pointed out a limited amino acid sequence similarity of p63 to sc-mtTFB. However, p63 later emerged to be a member of the large family of organellar PPR proteins and may rather be involved in posttranscriptional processes (Lurin, et al., 2004;Small and Peeters, 2000); J. Gualberto, IBMP CNRS, Strasbourg, France, personal communication).

The homology of nucleus-encoded plastid and mitochondrial RNA polymerases in plants and the presence of yet another phage-type RNA polymerase in both mitochondria and plastids in dicotyledonous plants (Chang, et al., 1999;Hedtke, et al., 1997;Hedtke, et al., 2000;Hedtke, et al., 2002;Ikeda and Gray, 1999), as well as the similarity of promoters that are recognized by these enzymes (Weihe and Börner, 1999) raise the question whether the two organelles harbour similar transcriptional cofactors interacting with these core RNA polymerases. The characterization of auxiliary factors in the plastid may well aid the identification of such proteins in the mitochondrion. CDF2, a DNA-binding factor isolated from spinach chloroplast, has been reported to stimulate transcription of the rrn operon by a nucleus-encoded RNA polymerase activity (Bligny, et al., 2000). However, structural details of CDF2 have hitherto escaped revelation (Bligny, et al., 2000), and no CDF2-like activity has been purified so far from plant mitochondria. Out of six nucleus-encoded sigma factors imported into Arabidopsis plastids, the protein designated Sig2 was found to additionally localize to mitochondria in GFP import assays (Tandara, 2000). Moreover, the maize orthologue Sig2B was determined by immunoblot analyses to co-purify with both mitochondria and plastids (Beardslee, et al., 2002). Yet, available experimental data so far relate Sig2 function to the bacterial-type plastidial RNA polymerase rather than phage-type enzymes in mitochondria or plastids (Beardslee, et al., 2002;Kanamaru and Tanaka, 2004), and references therein).

A regulatory role has recently been deduced of the plastid-encoded tRNA^Glu in plastidial transcription (Hanaoka, et al., 2005). Recombinant RpoTp specifically bound to the tRNA molecule in gel mobility shift experiments (Hanaoka, et al., 2005). Moreover, transcription from the plastidial accD promoter, which is considered to be catalyzed by a phage-type RNA polymerase, was shown to be inhibited by the addition of tRNA^Glu to in vitro transcription reactions using proplastid extracts from Arabidopsis as source of transcription activity (Hanaoka, et al., 2005). The authors suggested tRNA^Glu to mediate a switch in RNA polymerase utilization from nucleus-encoded RNA polymerases to the plastid-encoded bacterial-type enzyme during chloroplast development.