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- Materials and methods
We previously identified two multisubunit plastid RNA polymerases termed A and B. The B enzyme has a bacterial-type polypeptide composition and is sensitive to the prokaryotic transcription inhibitor rifampicin (Rif); the A enzyme has a more complex subunit structure and is Rif-resistant. Here we report results of N-terminal sequencing and MS carried out with the A enzyme, which establish that the latter contains rpo gene products and is structurally related to the B enzyme. Furthermore, evidence is provided that the A enzyme can be converted into a Rif-sensitive enzyme form in a phosphorylation-dependent manner in vitro by a treatment that results in depletion of a β-like subunit. Database searches using sequence information derived from additional polypeptides that are present in purified A preparations revealed sequence similarity with chloroplast proteins involved in RNA processing and redox control. This proteomics approach thus points to the complexity of the chloroplast transcription apparatus and its interconnections with post-transcriptional and signalling mechanisms.
Many metabolic functions of plants depend on light as an environmental signal. For instance, following exposure of dark-grown (etiolated) seedlings, they start greening and their cotyledons become converted into photosynthetic organs. At the subcellular level, this is reflected by the photoconversion of the nongreen plastids known as etioplasts into fully active chloroplasts, with accompanying changes in ultrastructure, protein composition, and gene expression patterns . Underlying control processes have mostly been assigned to the level of plastid RNA stability and translation [2,3], but accumulating evidence is available also for transcriptional control [4,5].
Recent progress in the analysis of the structure and function of the plastid transcription apparatus has revealed that at least two distinct DNA-dependent RNA polymerases are involved. One of them, which has long been known , is a multisubunit enzyme similar to those in bacteria and in the nuclei of eukaryotic cells. The other enzyme, which was detected more recently , is a single-subunit polymerase related to those of bacteriophages T3 and T7 and mitochondria [5,8,9]. The multisubunit enzyme was termed PEP (plastid-encoded polymerase) because of the intraorganellar coding sites of its core subunits, whereas the phage-type enzyme is nuclear-encoded [10–14] and hence was named NEP .
In mustard (Sinapis alba L.), we identified two distinct multisubunit plastid RNA polymerases, named enzymes A (cp-pol A) and B (cp-pol B), which differ in terms of subunit composition, functional properties, and abundance during etioplastchloroplast conversion . The type B RNA polymerase consists of four polypeptides (154, 120, 78 and 38 kDa) that match the predicted sizes of the plastid rpoC2, rpoB, rpoC1 and rpoA gene products from other plant species (reviewed in [16–18]). The type A enzyme is larger than the B enzyme, consisting of at least 13 putative subunits. Whereas the B enzyme is sensitive to the prokaryotic transcription inhibitor rifampicin (Rif) [19–21], the A enzyme is resistant to the drug. The latter enzyme form predominates in chloroplasts, whereas the B form is the major activity in etioplasts and in immature plastids during greening [15,22]. It was suggested, therefore, that the two enzyme forms may be structurally related, with a possibility of BA interconversion during chloroplast development .
To help clarify the possible structural and functional relationships of the A and B enzymes, two questions were addressed in the present work: (a) whether or not these two polymerases share common features at the level of the primary structure of their polypeptides; and (b) what might be the basis for their distinct properties, including Rif sensitivity vs. resistance.
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- Materials and methods
In this study we have obtained evidence, by N-terminal sequencing and MALDI-MS, for the existence of rpo-gene products in the multisubunit chloroplast RNA polymerase A from mustard. The N-terminal sequences of the two largest (141 and 110 kDa) polypeptides were found to match those of the derived rpoC2 and rpoB gene products (Fig. 3), which supports earlier suggestions that the chloroplast polymerase A is structurally related to the bacterial-type B enzyme that predominates in etioplasts [15,22]. In accordance with recent nomenclature , we propose to name these two enzyme forms PEP-A and PEP-B, respectively.
Microsequencing revealed that the N-terminal region of the 141-kDa PEP-A subunit from mustard is almost identical to that reported for the largest subunit of purified chloroplast RNA polymerase from maize, i.e. the only chloroplast RNA polymerase for which direct protein sequence information is as yet available [37,38]. On the other hand, the alignment of the derived protein sequences (Fig. 3) showed a short hypothetical extension, MEVL, for the dicot plant species mustard, Arabidopsis, spinach and tobacco, which is absent in the monocot species maize and rice. The perhaps most reasonable explanation would be that the second methionine residue in the dicot species marks the translation start site. However, it cannot be excluded that the extra peptide is synthesized and subsequently removed in vivo. It would be interesting to know if this region might be retained in RNA polymerase B from mustard. Despite various attempts, however, we consistently found the B subunits to be N-terminally blocked (data not shown).
The protein and DNA sequence data for the second largest (110 kDa) PEP-A subunit from mustard (Fig. 3) provide a picture that differs from that for the 141-kDa subunit. The microsequenced peptide aligns further upstream than that of the corresponding maize subunit , suggesting the existence of a 6 amino-acid extension in mustard. This is probably the result of the additional methionine residue in front of that used as the start methionine in most of the other plant sequences, except in the closely related crucifer Arabidopsis (Fig. 3).
The mass fingerprints of both the 110-kDa and 107-kDa polypeptides identified both as rpoB-related gene products. The presence of two forms of β subunit in PEP-A may seem peculiar, as in bacterial RNA polymerases β is the essential core subunit that catalyses polymerization of NTPs into RNA. An explanation suggesting a regulatory role comes from the rifampicin experiments (Fig. 4) discussed below. In the current work, we have not yet addressed experimentally the question of what might be the reason(s) for the different migration behaviour of these two sequence-related polypeptides, which could include proteolytic cleavage as well as a range of protein modifications. We note, however, that the 107-kDa but not the 110-kDa band was a substrate for PKA (Fig. 5), whereas neither of them was efficiently phosphorylated by PTK , suggesting that the different migration behaviour does not seem to simply reflect differences in phosphorylation state. While detailed analyses are part of our ongoing work, the present data obtained by microsequencing and MALDI-MS help to clarify another pertinent point. They exclude the possibility that either of these two bands might be closely related to the single-subunit NEP enzyme [13,14,39], which was reported to migrate in the 107–110-kDa region in spinach .
In addition to rpo gene products, microsequencing and MS identified three PEP-A components that are likely to represent nuclear gene products of diverse functions. The latter polypeptides, at 36 kDa (putative RNA binding protein), 29 kDa (annexin-like protein), and 26 kDa (Fe-SOD-like protein), all were consistently found in highly purified PEP-A preparations, including those that were subjected to two-dimensional gel electrophoretic separation (Fig. 2) or additional column chromatograpy steps. Furthermore, none of the determined sequences revealed contamination by major chloroplast proteins such as rubisco or components of the photosynthetic apparatus (data not shown), suggesting that the bands at 36, 29 and 26 kDa might be true constituents of chloroplast PEP-A.
What might be the functional role of these polymerase-associated components? The presence of a RNA binding protein (putative function of the 36-kDa component; Fig. 3) could be important in the stabilization and/or maturation of nascent transcripts. Both transcription and post-transcriptional RNA processing events are known to be driven by thylakoid-associated enzymatic machineries (reviewed in [1,40,41]). Moreover, evidence is available for components that act at the interface between transcription and RNA processing in a number of systems ranging from bacteria to vertebrates [42–44].
The existence of a Fe-SOD-related polypeptide (26-kDa band) in mustard PEP-A could be viewed as a consequence of thylakoid-associated chloroplast transcription. As photosynthetic electron transport can be a source of oxygen radicals [45,46], there is a need for efficient protection and detoxification mechanisms, in which SODs are known to play central roles [47,48]. One implication of the physical proximity therefore would be that newly synthesized transcripts must be protected from damage by radicals generated as photosynthetic by-products.
The annexin-like 29-kDa protein may have a related function. Plant annexins are calcium-dependent phospholipid-binding proteins that are often membrane-associated , which would be consistent with an anchoring role for this polymerase subunit. Furthermore, an Arabidopsis annexin that revealed significant similarity with the 29-kDa polypeptide was previously shown to counteract H2O2 stress in an oxyR-deficient Escherichia coli strain after transformation . This may point to a possible involvement of the 29-kDa protein in radical detoxification, i.e. as scavenger of H2O2 molecules generated by SODs, including the putative Fe-SOD (26-kDa band) of PEP-A (Fig. 3). Furthermore, H2O2 itself (or other reactive oxygen intermediates) might act as a transcriptional signal in chloroplasts, as was previously shown to be the case for oxyR-dependent transcription in bacterial systems [51,52]. We note that chloroplast transcription is modulated by redox-reactive reagents both in vitro[32,53] and in organello (T. Pfannschmidt and G. Link, unpublished data).
As shown in Figs 4 and 5, phosphorylation of PEP-A followed by phosphocellulose chromatography resulted in an enzyme form that had regained significant Rif sensitivity. The stained SDS/PAGE patterns (Fig. 5) revealed depletion of the 107-kDa (β107) subunit, indicating that this polypeptide may be a key determinant for Rif resistance vs. sensitivity. It is conceivable that the presence of this component affects the conformation of the PEP-A core in a way which prevents interference of Rif with transcription initiation, although the detailed mechanisms remain to be established. It seems reasonable to conclude, however, that the phosphorylation/phosphocellulose treatment used (Fig. 4) does not result in an altered phosphorylation state of the β107 component itself. It is not an efficient substrate for PTK, i.e. the endogenous serine-type protein kinase used for the pretreatment of PEP-A . The loss of the β107 subunit thus seems to be an indirect effect in reponse to phosphorylation of other PEP-A polypeptide(s), the most likely candidates of which are the known PTK substrate bands at 72–76 kDa .
In terms of our model proposed for the BA conversion during light-induced chloroplast formation [15,23], the ‘nonrpo’ polypeptides could be viewed as AAFs (PEP-A-associated factors) that are recruited into the large A enzyme complex. These AAFs provide new functions to the transcriptional machinery, which may help to adapt and regulate transcription under conditions of increasing photosynthetic activity. Our data support the current view that the chloroplast transcription apparatus is much more complex than could be envisaged from the bacterial-type organization of the rpo genes, which fully account for the α(2)ββ′β″ architecture of the etioplast B enzyme.
Both the number and composition of accessory polypeptides of the transcription complex seem to vary depending on age, tissue type and environmental cues. For instance, different forms of chloroplast RNA polymerase complexes were purified from pea, each of which contained both common and distinct polypeptides and revealed different transcriptional properties . Likewise, the PEP enzyme from wheat was recently reported to show tissue type-specific transcription specificity . Control mechanisms that regulate the recruitment and activity of the polymerase-associated factors are beginning to be elucidated. Further characterization of these factors, preferably in a cloned recombinant form, can be anticipated to help clarify their role during plastid development and function.