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Genes to Cells (2004) 9, 1175-1187. doi:10.1111/j.1365-2443.2004.00808.x
© 2004 Blackwell Publishing or its licensors
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In vitro transcription analysis by reconstituted cyanobacterial RNA polymerase: roles of group 1 and 2 sigma factors and a core subunit, RpoC2

Sousuke Imamura, Munehiko Asayama* and Makoto Shirai

Laboratory of Molecular Genetics, College of Agriculture, Ibaraki University, Ami, Inashiki, Ibaraki 300-0393, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
The RNA polymerase (RNAP) core enzyme of cyanobacterium Synechocystis sp. strain PCC 6803 was reconstituted with overproduced recombinant subunits and purified with C-terminal histidine-tagged RpoA. The core enzyme with purified a sigma factor, SigA/SigD or SigB, allowed specific in vitro transcription from the light-inducible psbA2 or the dark-/heat-inducible lrtA/hspA promoters, respectively. Further analysis using a mutant psbA2 promoter revealed that the –35 hexamer of the promoter was essential for SigA but not SigD. Similar but distinct patterns of psbA2 transcription were found for two types of RNAP, cyanobacterial ({alpha}2ßß'{gamma}) and E. coli ({alpha}2ßß') core enzymes. Specific binding of PCC 6803 RpoC2 (ß') to E. coli core enzyme and its contribution to efficient psbA2 transcription by RNAP-SigA/D suggest that this subunit could confer an important role on the cyanobactrial RNAP. Differences in affinity and specificity among cyanobacterial sigma factors for the core enzyme and promoters were discussed.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
The RNA polymerase (RNAP) of eubacteria consists of a core enzyme and a sigma factor. The core enzyme is capable of transcription elongation, whereas the sigma factor is required for specific promoter recognition and transcription initiation (Ishihama 1993, 2000). So, the sigma factor plays a key role in the initial step of transcription. The RNAPs of cyanobacteria, which can perform oxygen-evolving photosynthesis, have been known to be closely related to the eubacterial RNAPs. Although most eubacterial RNA polymerase holoenzymes consist of a core enzyme with subunits of {alpha}2ßß' and one of several species of sigma factors (Ishihama 1993), the ß' subunit is usually split into two parts in cyanobacteria, {gamma} (RpoC1) and ß' (RpoC2), the same as in higher plant chloroplasts. Thus, the RNAP holoenzyme of cyanobacteria consists of a core enzyme with the subunit structure {alpha}2ßß'{gamma} and one of the species of sigma factor (Schneider et al. 1987). The eubacterial ß' (RpoC) subunit, for example of Escherichia coli, possesses eight highly conserved domains, A to H (Allison et al. 1985). In cyanobacteria and also plant chloroplasts, RpoC1 and RpoC2 possess the domains A to D and E to H, respectively (Schneider & Haselkorn 1988; Bergsland & Haselkorn 1991).

In a cyanobacterium Synechocystis sp. strain PCC 6803, nine sigma factors (group 1, SigA; group 2, SigB to SigE; group 3, SigF to SigI) are identified and their functions have been tentatively assigned in some cases. As examples, SigB is induced under heat-shock and dark conditions in which the electron transport chain of photosynthesis is in an oxidative state. This sigma factor contributes to the transcription of heat-inducible hspA or dark-inducible lrtA genes (Imamura et al. 2003a,b). SigD is antagonistically induced under light/high-light conditions in which the electron transport chain of photosynthesis is in a reductive state. This sigma factor contributes to the transcription of photosynthesis genes encoding the D1 protein in photosystem II (PSII), psbA2 and psbA3 (Imamura et al. 2003a,b). Furthermore, SigC contributes to global gene expression, involving the nitrogen-related glnB gene, in the phase of post-exponential cell growth (Asayama et al. 2004). SigE might also contribute to the transcription of nitrogen-related genes, such as glnN (Muro-Pastor et al. 2001).

In vitro transcription system with purified RNAP is an essential tool for biological characterization of the promoter selectivity of sigma factors. In cyanobacteria, RNAP core enzyme has been purified from Anabaena and Synechococcus cells, and in vitro transcription systems were established (Schneider et al. 1987; Goto-Seki et al. 1999). We also tried to purify RNAP from the Synechocystis PCC 6803 cells. Although polyethyleneimine (PEI) precipitation for RNAP purification was useful in E. coli and some cyanobacteria (Burgess & Jendrisak 1975; Schneider et al. 1987; Goto-Seki et al. 1999), the product with PEI was seriously aggregated in the case of PCC 6803. This problem has not been yet finally solved. Therefore we have used heterologous RNAP holoenzymes reconstituted with the E. coli core enzyme and PCC 6803 sigma factors for in vitro transcription analysis to date (Imamura et al. 2003b). The cyanobacterial and eubacterial (E. coli)-type core enzymes are essentially the same in molecular structure and function, however, differences in transcription have been observed between them even with the same promoter (Schneider et al. 1987; Imashimizu et al. 2003; Ito et al. 2003). Consequently, an easy purification procedure of PCC 6803 core enzyme was required for in vitro transcription system with homologous RNAP subunits. In this paper, we report the establishment of the in vitro transcription system using reconstituted RNAPs, composed of purified recombinant subunits as the first such case in photosynthesizing organisms. This in vitro system was useful for characterization of cyanobacterial promoters. Roles of cyanobacterial sigma factors and RpoC2 were also revealed.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 Supplementary material
 References
 
Purification of reconstituted PCC 6803 RNAP core enzyme

To establish an in vitro transcription system, we initially tried to purify the core enzyme from PCC 6803 cells. However, this proved too difficult (see Introduction). Therefore, each RNAP core subunit was overproduced in E. coli in an attempt to reconstitute them in vitro. To avoid contamination of E. coli RNAP subunits, the recombinant PCC 6803 core subunits were recovered from inclusion bodies as an insoluble fraction. In the case of PCC 6803 RpoA, the 6xHis-tag was attached in the C-terminal region (RpoA-His). This RpoA-His was homogeneously purified by an affinity column chromatography (Ni2+-nitrilotriacetic acid (Ni-NTA)) under denaturing condition with 6 M GuHCl (guanidine hydrochloride) or 8 M urea (data not shown). On the other hand, RpoB, RpoC1 and RpoC2 possess no His-tag. Their enzymes were roughly purified to > 70% homogeneity from the inclusion bodies in the first step (see Experimental procedures). Fractions containing the four subunits were mixed together and dialysed against the reconstitution buffer. The reconstituted PCC 6803 RNAP core enzyme was again purified through the Ni-NTA column. The purified core enzyme was resolved on an SDS-PAGE gel (Fig. 1A) and its quality was verified by Western blotting with polyclonal antibodies against PCC 6803 core subunits (Imamura et al. 2003b) (Fig. 1B, upper panel). Each PCC 6803 core subunit in the purified fraction was well recognized by respective antibodies. A signal corresponding to slightly larger molecular weight of RpoA-His in the purified PCC 6803 core fraction compared with that of authentic RpoA in the PCC 6803 cell was observed. Of note, PCC 6803 RpoA and RpoB antibodies crossreacted to E. coli RpoA and RpoB subunits, respectively, in this condition. PCC 6803 SigA and E. coli RpoB antibodies also could recognize E. coli RpoD ({sigma}70, Sigma70) and PCC 6803 RpoB, respectively (Fig. 1B, lower panel). We could finally exclude the possibility of contamination with E. coli RNAP core subunits and RpoD in the PCC 6803 core enzyme fraction (Fig. 1B, lower panel). Thus, the reconstituted PCC 6803 core enzyme was successfully prepared. This purified core enzyme and the purified His-tagged sigma factor were mixed, again reconstituted for holoenzymes, and subjected to in vitro assay.



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  		Figure 1  Purified RNAP core enzyme. (A) Purified E. coli (lane 1) and PCC 6803 (lane 2) core enzyme. The samples were resolved by 10% SDS-PAGE (0.2 µg each). The gel is stained with Coomassie Brilliant Blue. The positions of E. coli{alpha} (RpoA, 37 kDa), ß (RpoB, 151 kDa), and ß' (RpoC, 155 kDa) and PCC 6803 {alpha}-His (RpoA-His, 39 + 1 = 40 kDa), ß (RpoB, 129 kDa), ß' (RpoC2, 159 kDa), and {gamma} (RpoC1, 65 kDa) are indicated by arrowheads, respectively. (B) Confirmation of PCC 6803 core enzyme. Purified E. coli core or holoenzyme (0.2 µg or 0.01 µg), purified PCC 6803 core enzyme (0.2 µg) and PCC 6803 total cellular proteins (10 µg) prepared from a mid-log phase were resolved by 10% SDS-PAGE. Then, they were subjected to Western blotting (Imamura et al. 2003a, b) with relevant antibodies. The presence (+) and absence (–) of proteins are indicated at the top. Asterisks indicate the positions of relevant band for antibodies.

 
Enzyme activity of RNAP and optimal conditions

To test the activity for mRNA synthesis of the reconstituted RNAPs, we performed in vitro run-off transcription assays with the PCC 6803 psbA2 promoter having a canonical E. coli consensus promoter. Supercoiled plasmids (Table 1) and the reconstituted cyanobacterial RNAP holoenzymes were used in the assay. As shown in Fig. 2A, significant mRNA products were obtained with RNAPs of PCC 6803 core enzyme +SigA (lane 4) as well as E. coli core enzyme +SigA (lane 2). No product was detected using only the PCC 6803 core enzyme or the negative control fraction (lanes 3 and 5). These results clearly show that the PCC 6803 core enzyme is not contaminated with E. coli RNAP subunits, and the reconstituted PCC 6803 RNAP has enzyme activity in vitro. We also assessed the optimal conditions for transcription by the RNAP. Effective transcription was observed at a temperature, pH, and concentration of MgCl2, of 30–50 °C, 7.0–8.0, and 5 mM, respectively (Fig. 2B–D). Thus we did the run-off assay in vitro under optimal conditions with a reaction solution comprised of 50 mM Tris-HCl (pH 7.9), 5 mM MgCl2, 0.05 mM EDTA·2Na, 0.5 mM DTT, 0.1 mM each of ATP/CTP/GTP, 0.05 mM[{alpha}-32P] UTP (18.5 GBq/mmol), 25 nM RNAP core enzyme, 100 nM sigma factor, and 2.5 nM various template DNA at 30 °C.


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  		Table 1  Template DNAs and promoters
 


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  		Figure 2  RNAP enzyme activity and optimal condition for in vitro transcription. (A) In vitro run-off transcription analyses with pKK-A2 (PCC 6803 psbA2 promoter, Table 1) and RNAP. The presence (+) or absence (–) of the E. coli core enzyme (Fig. 1A, lane 1), purified protein as a negative control (RpoA-His +an E. coli negative fraction, see Experimental procedures), reconstituted PCC 6803 core enzyme (Fig. 1A, lane 2), and PCC 6803 SigA is indicated at the top. Arrowhead indicates the position of the product (329 nt) synthesized from the psbA2 promoter. The positions of molecular size markers, 32P-end-labelled fragments derived from pUC119B DNA digested with HinfI (Imamura et al. 2003b), are presented in nucleotides (nt) at the left. (B–D) Effects of temperature (B), pH (C), and MgCl2 (D) on transcription from the psbA2 promoter. Each reaction was done with RNAP composed of the PCC 6803 core enzyme and SigA (panel A, lane 4). The profiles of synthesized mRNA are shown as insets. Signal intensities on X-ray films from three independent experiments were quantified as previously described (see previous data: Imamura et al. 2003b) and the values are presented (n = 3, means ± Standard Deviation) as relative levels (the highest value is taken as 100%).

 
C-terminal His-tagged SigB

Previous studies in vitro showed transcription by RNAPs with PCC 6803 group 1 (SigA) and group 2 (SigB, SigC, SigD, SigE) sigma factors, which are His-tagged at the N-terminal except for SigC. However, the rate of transcription by RNAP with SigB seemed to be relatively low (Imamura et al. 2003b). This might be caused by low enzyme activities for SigB if the tag is fused at the N-terminal (His-SigB). Therefore, we again tried to construct a C-terminal His-tagged SigB (SigB-His). Although the level of SigB-His from the expression vector was very low in E. coli, compared with that of His-SigB, we eventually succeeded in purifying SigB-His (data not shown). As shown in Fig. 5, apparent mRNA products were more effectively synthesized by RNAP with SigB-His (Fig. 5A) than His-SigB (see previous data: Imamura et al. 2003b). This purified SigB-His was therefore used.



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  		Figure 5  Similar but distinctive effects on transcription of each RNAP composed of the E. coli or PCC 6803 core enzyme. (A) In vitro run-off transcription assay with the PCC 6803 psbA2 promoter (pKK-A2). The heterologous (E. coli core +PCC 6803 Sig'X', left panel) or homologous (PCC 6803 core +PCC 6803 Sig'X', right panel) RNAPs were used. Others are the same as in Fig. 2A. (B) Relative levels of the transcripts. Signal intensities on X-ray films from three independent experiments were quantified the same as in Fig. 2 and the values are presented (n = 3, means ± SD) as relative levels (value for SigA as 100%).

 
Promoter selectivity of group 1 and 2 sigma factors

E. coli possesses only one group 2 sigma factor, RpoS ({sigma}S, Sigma38). Although the existence of multiple group 2 sigma factors is a characteristic of cyanobacteria, the individual promoter selectivity among them remains to be resolved. The promoter recognition specificity of the group 2 sigma factors was examined in vitro with heat-shock hspA and dark-induced lrtA promoters, which are specifically recognized in vivo by SigB under conditions of an oxidized electron transport chain of photosynthesis in PCC 6803 cells (Imamura et al. 2003a,b), under the optimal conditions. The results are shown in Fig. 3. The supercoiled DNA templates for each promoter were summarized in Table 1. The hspA promoter is the E. coli consensus-type promoter except for a short spacer length (15-bp). On the other hand, lrtA possesses only the –10 hexamer. The signal intensities from the psbA2 promoters were relatively greater than those of hspA and lrtA for each sigma factor, indicating that psbA2 has a strong promoter. Both the hspA and lrtA promoter were well recognized by SigB (Fig. 3A,B). For high-resolution analysis on hspA and lrtA transcription, we further performed primer extension following in vitro mRNA synthesis (Fig. 3D). The result was not contradict to that shown in Fig. 3A. These well support the data obtained in vivo (Imamura et al. 2003a,b). These results also show the promoter recognition specificity in vitro with redundancy among group 2 sigma factors but with differences in efficiency. The differences in specificity might depend on the promoter architecture and potential ability of each sigma factor, since no other trans-acting factor was added into the assay. We confirmed almost the same signal intensities of transcripts from the RNA I promoter in each plasmid, as a control shown in Fig. 3C.



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  		Figure 3  Promoter selectivity of group 1 and 2 sigma factors. (A) In vitro run-off transcription assay of the PCC 6803 psbA2 (pKK-A2), hspA (pKK-PA), and lrtA (pKK-TA) promoters. The reconstituted PCC 6803 RNAPs with SigA to SigE were used. Parts of each synthesized mRNA on an X-ray film are shown. (B) Relative levels of the transcripts. Signal intensities on the X-ray films from three independent experiments were quantified the same as in Fig. 2. The values (each value of psbA2 as 100%) were presented (n = 3, means ± S.D.). (C) Relative levels of the transcripts from RNA I promoter on pKK-A2, pKK-PA and pKK-TA. The transcripts synthesized by the reconstituted PCC 6803 RNAP with SigA served as a control. A profile of the transcripts from RNA I promoter is shown as an inset. (D) High resolution analysis for hspA and lrtA transcription. The hspA and lrtA transcripts were synthesized in vitro in a mixture comprising of PCC 6803 core enzyme (2 pmol), each sigma (6 pmol) and supercoiled template DNAs (pKK-PA or pKK-TA, 0.3 pmol). Then, primer extension (Asayama et al. 2004) was done with sll1514-R (Imamura et al. 2003b) or lrtA-R4 primer (Imamura et al. 2003a), respectively. The transcription start points (–44 or –312, +1 as the initiation codon) are shown together with analysed with total RNA prepared from the PCC 6803 cells as a positive control.

 
Effect of –35 hexamer on promoter recognition

The major class of bacterial promoters is defined by two conserved elements, the –10 and –35 hexamers that are in contact with sigma factors in the RNAP holoenzyme. Amino acid sequences for promoter recognition are similar but not identical between RpoD (group 1) and RpoS (group 2) proteins in E. coli. Although the –10 hexamer was conserved in cases of both sigma factors, RpoS promoters have less conservation at the –35 hexamer (Tanaka et al. 1993; Espinosa-Urgel et al. 1996). To address the influence of the –35 hexamer sequence for group 1 and group 2 sigma factors, mRNA synthesis in vitro was examined with SigA and SigD (Fig. 4), which take part in psbA transcription in PCC 6803 cells (Imamura et al. 2003a,b). The psbA2 promoters (Table 1) in pKK-IY1 (wild-type, –35 and –10 hexamers) and pKK-IY4 (–10 hexamer only, AC->TG mutations at the –35 hexamer) were used and derived from another cyanobacterium M. aeruginosa K-81 (Ito et al. 2003), which is categorized into the same group of Synechocystis species. In promoter architecture, K-81 psbA2 and PCC 6803 psbA2 are essentially the same (Shibato et al. 1998, 2002). Results indicated that the –35 hexamer sequence is essential for SigA but not for SigD, suggesting a difference in the requirement of a conserved –35 hexamer for promoter recognition between the group 1 and group 2 sigma factors. We also confirmed equal amounts of mRNA products from the RNA I promoter on pKK-IY1 or pKK-IY4 as a control (Fig. 4B).



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  		Figure 4  Effects of the –35 hexamer on promoter recognition. (A) In vitro run-off transcription assay with template DNAs of pKK-IY1 [WT, wild-type K-81 psbA2 (–35 & –10) and pKK–IY4 [–35mt, K-81 psbA2 promoter (–10 only)]. The reconstituted PCC 6803 RNAPs with SigA or SigD were used. Signals corresponding to the K-81 psbA2 transcripts on X-ray film are presented. (B) Relative levels of the transcripts. Signal intensities of transcripts from psbA2 or RNA I promoter on X-ray films from three independent experiments were quantified the same as in Fig. 2. The values (each value of pKK-IY1 as 100%) are presented (n = 3, means ± SD) as relative levels.

 
Distinctive effects on transcription of each RNAP composed of the E. coli or PCC 6803 core enzyme

The subunit composition of the core enzyme in photosynthesizing organisms is distinct from that in non-photosynthesizing eubacteria (Introduction). Therefore, we compared the RNAP activities between PCC 6803 core ({alpha}ßß'{gamma}) and E. coli core ({alpha}ßß') enzymes on the PCC 6803 psbA2 transcription. Similar but distinct signal patterns were observed in the gel profile even when the same sigma factor was used (Fig. 5A, left vs. right). Interestingly, the PCC 6803 RNAP holoenzyme, E{sigma}D, composed of the PCC 6803 core enzyme (E) and SigD ({sigma}D), drove the transcription more than did the RNAP with E. coli core enzyme (Ec) (lane 4). In contrast, the signal transcribed by E{sigma}C (PCC 6803 core +SigC) was significantly weaker than that transcribed by Ec{sigma}C (E. coli core +SigC) on the promoter (lane 3). The psbA2 promoter is specifically recognized by light/high-light-induced SigD in PCC 6803 cells (Imamura et al. 2003a, b). The in vitro results coincide well with previous data obtained in vivo. These findings indicated that the functions of cyanobacterial and E. coli core enzymes are similar but distinct. This idea led us to perform the next experiment on the characterization of the core enzyme.

Binding of RpoC1 and RpoC2 to the E. coli core enzyme and role of PCC 6803 RpoC2 in transcription

E. coli-type ß' (RpoC) refers to two parts of {gamma} (RpoC1) and ß' (RpoC2) in cyanobacterial RNAP (Fig. S1A). A phylogenetic analysis was therefore performed on RpoC1 or RpoC2 together with the corresponding segments of E. coli-type RpoC (Fig. S1B). In both cases, four groups were constituted as Gram-positive or -negative non-photosynthesizing bacteria, cyanobacteria and plants. The molecular structure of RpoC2 has devolved more extensively than RpoC1 subunits. This suggests that RpoC2 is evolutionarily a unique core subunit. We therefore investigated the contribution of cyanobacterial RpoC2 to transcription. If RpoC2 plays some unique role, a heterologous RNAP holoenzyme composed of the E. coli core enzyme and PCC 6803 sigma factor (Ec{sigma}) will be able to enhance its ability specifically. First, we assessed the binding affinity of PCC 6803 RpoC2 for the E. coli core enzyme (Ec) in a His-tag pull-down assay. We also used PCC 6803 RpoC1 as a control. As shown in Fig. 6, the E. coli RpoA, RpoB and RpoC bands were only detected when the E. coli core was mixed with His-RpoC1 or His-RpoC2 (lanes 3, 6 and 9 in panel A and lanes 3, 12 and 15 in panel B). The band intensities of RpoA were not altered by addition of bovine serum albumin (BSA) (lane 3 vs. lanes 4–6 in panel B) or {alpha}-casein as a competitor (lane 3 vs. lanes 7–9 in panel B), indicating the specific-binding of the E. coli core enzyme to His-RpoC2. These results showed that RpoC1 and RpoC2 are capable of binding to the E. coli core enzyme complex. Second, the affinity of RpoC2 in RNAP (Ec +RpoC2) for PCC 6803 sigma factors ({sigma}A, {sigma}C and {sigma}D) was tested using PCC 6803 psbA2 transcription (Fig. 7). The signal intensities in Fig. 7A–C are summarized in Fig. 7D. It was of great interest that the transcription from psbA2 was markedly accelerated by a combination of RpoC2 and SigD (Ec{sigma}D+RpoC2) (panel C, lanes 9 vs. 10 and 11) even under conditions in which the amount of SigD relative to E. coli core enzyme is saturated (lanes 2–5). Similar results by RNAP (Ec{sigma}D+RpoC2) were also observed on the RNA I promoter carrying an E. coli consensus-type sequence as well as that of the psbA2 promoter (Goto-Seki et al. 1999). In contrast, no enhancement was observed on addition of RpoC1 (panel C, lane 6 vs. 7 and 8). In addition, a combination of RpoC2 and SigA in the RNAP (Ec{sigma}A+RpoC2) also enhanced the transcription (panel A, lanes 4 vs. 5 and 6) but not so another RNAP (Ec{sigma}C+RpoC2) containing RpoC2 and SigC (panel B, lanes 4 vs. 5 and 6). We also checked whether the amount of SigC (5 pmol) and SigA (5 pmol) is also saturated (data not shown). No enhancement by RpoC2 was observed when the PCC 6803 core enzyme was used the same as the E. coli core (data not shown). These results suggest that cyanobacterial RpoC2 plays a role in the enhancement of transcription. This is the first time that a specific enhancement of transcription by a group 2 sigma factor has been confirmed directly in vitro on a light-responsive promoter in photosynthesizing organisms.



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  		Figure 6  Binding affinity of PCC 6803 RpoC1 and RpoC2 to E. coli core enzyme. (A) His-tag pull-down assay with PCC 6803 His-RpoC1 and E. coli core enzyme. The presence (+) and absence (–) is indicated at the top. Details are described in Experimental procedures. The specific binding of the E. coli core enzyme to PCC 6803 RpoC1 was detected by Western blotting with E. coli RpoA, RpoB, and RpoC antibodies. Asterisks indicate the positions of relevant band. (B) The assay with PCC 6803 His-RpoC2 and E. coli core enzyme. Others are the same as (A).

 


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  		Figure 7  Role of RpoC2 in cyanobacterial RNAP in transcription. (A–C) In vitro run-off transcription assay was done with the template pKK-A2 (psbA2 and RNA I promoters) and SigA (A), SigC (B), or SigD (C). The amount of enzymes (pmol) is shown at the top. In the case of addition of PCC 6803 RpoC1 or RpoC2, PCC 6803 RpoC1 or RpoC2 and the E. coli core enzyme were incubated at 30 °C for 30 min the same as in Fig. 6 before the assay. The transcripts from psbA2 and RNA I promoters are indicated by arrowheads. (D) Relative ratios of the transcripts with RpoC1 or RpoC2. Signal intensities on X-ray films from three independent experiments were quantified the same as in Fig. 2 and the relative ratio (presence of RpoC1 or RpoC2 (1 pmol)/absence) is presented (n = 3, means ± SD).

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 Supplementary material
 References
 
An in vitro transcription system using homologous reconstituted RNAP with recombinant subunits was tried for the first time in photosynthesizing organisms. The in vitro results coincide well with previous in vivo data (Imamura et al. 2003a,b), indicating that this in vitro system is useful for transcription analysis. The ineffective step of PEI precipitation was also observed in the case of M. aeruginosa K-81 RNAP (Asayama et al. 1996), suggesting that the PEI precipitation might not be useful for RNAP in Synechocystis species. Thus the approach in this study will shed light on in vitro transcription analyses for RNAPs not only of cyanobacteria but also of other photosynthesizing organisms. In addition, this in vitro assay system will be also convenient to functional analyses with mutant RNAPs even if the mutants cause to lethal cells. Of note, the C-terminal His-tagged RpoA (RpoA-His) was useful for purification of the reconstituted RNAP core enzyme in this study. The N-terminal domain of RpoA ({alpha}-NTD) is known to play a key role in assembly of the core enzyme (Igarashi et al. 1991). Tang et al. (1995) have demonstrated that E. coli RpoA-His potentially functions normally on the CAP-dependent lacP (ICAP) promoter, although the C-terminal domain of RpoA ({alpha}-CTD) is required for efficient transcription with CAP.

Here, we discuss the specific promoter selectivity and roles of PCC 6803 group 1 and 2 sigma factors as follows. In the case of SigA, both the –35 and –10 hexamers are needed for efficient transcription, the same as with E. coli RpoD (Figs 3–5). The specific promoter selectivity of SigB was directly confirmed in vitro with redundancy of other sigma factors on the heat-shock hspA or dark-inducible lrtA promoter (Fig. 3). The spacer of the hspA promoter is short, and the lrtA promoter has no conserved –35 hexamer (–10 only). This suggests that SigB may be a sigma factor capable of recognizing non-consensus-type promoters. The lrtA promoter has TGTGn and GC motifs (Table 1, dots and *, respectively), which are directly recognized by region 2.5 of E. coli RpoD and RpoS, respectively (Barne et al. 1997; Burr et al. 2000; Becker & Hengge-Aronis 2001). Therefore, these motifs may have some effect on promoter recognition by SigA and SigB in PCC 6803 cells. Recent experiments revealed that Hik34, a histidine kinase protein of a two-component regulatory system, regulates hspA transcription (Marin et al. 2003), suggesting that a set of regulatory factors also participates in hspA expression in cooperation with the sigma factors. In the present study, C-terminal His-tagged SigB was used and its ability improved in vitro compared with a previous report (Imamura et al. 2003b) (Figs 3 and 5). The reason for the inability of the N-terminal His-tagged SigB to act is not clear. The proportion (approximately 60%) of hydrophobic amino acids upstream of region 1.2 in the N-terminal SigB is the highest among PCC 6803 group 1 and 2 sigma factors. SigC seems to prefer both –10 and –35 elements, the same as SigA (Fig. 3). However, recent in vivo and in vitro studies revealed that SigC specifically recognized the glnB P2 promoter, lacking the –35 hexamer, under nitrogen starvation conditions during the post-exponential growth phase (Asayama et al. 2004). In this case, the function of the –35 sequence may be rescued by the binding of NtcA (a transcription factor of the CRP-family) to the region. In future, the point of the promoter selectivity of SigC in two types (–35/–10 or –35 lacking but with a trans-acting factor) of promoter will be solved in vitro. In the case of SigD, a flexible type of promoter recognition was observed (Figs 3 and 4). Light/high-light-induced SigD can specifically recognize the psbA2 promoter in redundancy with SigA under conditions of reductive stress in PCC 6803 cells (Imamura et al. 2003a,b). Our previous studies also showed that the SigA type sigma factor can specifically recognize the psbA2 promoter (Shibato et al. 1998, 2002; Ito et al. 2003). Taking all these findings into considerations, the PCC 6803 psbA2 promoter might be constitutively recognized by SigA, but there could be a ‘switch’ from SigA to SigD in RNAP at least under the light/high-light conditions. How can SigD take advantage of the situation? Recently, it has been indicated that sigma factor competition toward the limited core enzyme, which is regulated by the sigma factor's expression levels, degradation rate, anti-sigma factor, or guanosine-3,5-(bis)pyrophosphate (ppGpp), is an important aspect of group 2 and 3 sigma factor-dependent transcription (Muffler et al. 1997; Farewell et al. 1998; Jishage & Ishihama 1999; Maeda et al. 2000; Zhou et al. 2001; Jishage et al. 2002). In the case of SigD on the psbA2 promoter, it is conceivable that the switch may be caused by an increase in the SigD protein level. The level was significantly induced during light/high-light conditions under which absolute intracellular levels are almost the same for major sigma factor, SigA (approximately 8 fmol/µg total protein) (Imamura et al. 2003a,b). Alternatively, it is possible that a regulatory system operates through the –35 hexamer sequence with a trans-acting factor(s), which can specifically bind to the region and affect the promoter recognition by SigA and SigD for which the requirement for conserved –35 hexamer was quite different (Fig. 4). Our previous studies have also indicated that the –35 hexamer of the K-81 psbA promoter is indispensable for light-dependent and circadian transcription in cyanobacterial cells (Shibato et al. 1998, 2002; Ito et al. 2003).

Two core subunits of {gamma} (RpoC1) and ß' (RpoC2) are unique in photosynthesizing organisms and distinct from those of other eubacteria (Schneider et al. 1987; Xie et al. 1989). Immunological studies have indicated that the E. coliß' (RpoC) subunit is closely related to cyanobacterial {gamma} (RpoC1) subunit but not to ß' (RpoC2) (Schneider & Haselkorn 1988). This supports the result of phylogenetic analysis that RpoC2 subunits have evolved more extensively than RpoC1 subunits. The roles of cyanobacterial {gamma} (RpoC1) and ß' (RpoC2) were characterized in vitro for the first time in this study. Although both subunits are capable of binding the E. coli core enzyme, RNAP with PCC 6803 RpoC2 could only accelerate the transcription on the psbA2 promoter (Fig. 7). The RNAP with SigD could drive a more effective transcription with the reconstituted core enzyme of PCC 6803 than E. coli (Fig. 5). These results suggest that RpoC2 caused a structural change in the E. coli core enzyme to adopt the cyanobacterial form. Then, the binding affinity of sigma factors for the core enzyme and/or potential ability of the sigma factors in RNAP holoenzyme might be enhanced.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
Supplementary material
 References
 
Expression vectors and overproduction of RNAP core subunits

For the construction of expression vectors, each DNA fragment containing the RNAP core subunit structural gene was amplified by PCR with PCC 6803 templates previously described (Imamura et al. 2003b) and a set of primers (Table 2). Each primer included a unique restriction enzyme site (underlined in the sequences shown in Table 2). The PCR-amplified fragments were digested with appropriate restriction enzymes and the resultant fragments were then cloned into the corresponding sites of pQE70 (QIAGEN) or pKK223-3 (Amersham Pharmacia Biotech) to create the expression vectors, pQE1818A70, pQE1787B(-His), pKK1265G and pKK1789C for the expression of a C-terminal histidine hexamer-tagged (His-tag) RpoA, RpoB, RpoC1 and RpoC2 in E. coli, respectively. To over-express each RNAP core subunit gene, E. coli strain M15[pREP4] containing pQE1818A70 or pQE1787B(-His) or E. coli strain JM105 containing pKK1265G or pKK1789C, was cultivated at 37 °C in 250 mL of 2xTY medium (Ish-Horowicz & Burke 1981) containing ampicillin (75 µg/mL) and kanamycin (15 µg/mL) or ampicillin (75 µg/mL), respectively. When the turbidity of the cell culture reached 0.5 absorbance units at A660, IPTG (isopropyl-ß-D-thiogalactopyranoside) was added at a final concentration of 2.0 mM. After incubation at 37 °C for 5 h, the cells were harvested by centrifugation and stored at –80 °C prior to use. For the negative control experiment, E. coli strain M15[pREP4] containing pQE70 or strain JM105 containing pKK223-3 was treated as mentioned above.


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  		Table 2  Primers used for amplification
 
Purification of reconstituted RNAP core enzyme

Reconstitution and purification of the RNAP core enzyme were performed basically as described for E. coli RNAP holoenzyme (Tang et al. 1995), but with some modifications. After thawing on ice, the cells were suspended in 15 mL of lysis buffer (Imamura et al. 2003b) and disrupted by sonication (model 250D, Branson). After centrifugation, each over-expressed gene product was recovered as inclusion bodies. The purification of His-tagged RpoA under denaturing conditions was performed as previously described (Imamura et al. 2003b). The eluted recombinant RpoA-His was precipitated with ammonium sulphate at a final concentration of 70% and recovered by centrifugation. The pellet was dissolved in 1 mL of Buffer G (6 M GuHCl, 50 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 10 µM ZnCl2, 1 mM EDTA·2Na, 10 mM dithiothreitol and 10% glycerol). For the preparation of RpoB, RpoC1 and RpoC2, the inclusion bodies were directly dissolved in 5 mL of Buffer G. After clearance of the debris by centrifugation, each supernatant was mixed in the following molar ratio, {alpha}:ß:ß':{gamma} = 1 : 1 : 2 : 4, to reconstitute the PCC 6803 core enzyme. For the negative control experiment, the purified RpoA-His obtained from the Ni-NTA column was mixed with the same amount of the extracts obtained from the relevant E. coli strain containing only expression vector, pQE70, or pKK223-3 (Fig. 2A, lane 5). The mixtures were then dialysed against the reconstitution buffer (50 mM Tris-HCl (pH 7.9), 200 mM KCl, 10 mM MgCl2, 10 µM ZnCl2, 1 mM EDTA·2Na, 5 mM 2-mercaptoethanol and 50% glycerol) to reconstitute PCC 6803 core enzyme for 16 h at 30 °C. After centrifugation, the supernatant was collected and applied to a Ni-NTA column at 4 °C (QIAGEN, column volume 3 mL, 14 mL/h) equilibrated in TG10–50 buffer (10 mM Tris-HCl (pH 7.9), and 50% glycerol) containing 5 mM imidazole. The column was washed with 10 mL of TG10–50 buffer containing 5 mM imidazole and 50 mL (10 mL, 5 times) of TG10–50 buffer containing 20 mM imidazole. The reconstituted core enzyme complexes were eluted with 20 mL of TG10–50 buffer containing 250 mM imidazole. Fractions containing the core enzyme were confirmed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and dialysed against storage buffer, comprised of 50 mM Tris-HCl (pH 7.9) 50% glycerol, 0.1 mM EDTA·2Na, and 0.1 mM dithiothreitol, and stored at –80 °C.

Preparation of C-terminal His-tagged SigB

For the construction of SigB-His, a DNA fragment containing the sigB structural gene was amplified by PCR with PCC 6803 genomic DNA as a template and a set of primers (Table 2). The PCR-amplified fragment was digested with SphI and BglII and the resultant fragment was then cloned into the corresponding sites of pQE70 (QIAGEN) to create the expression vector pQE0306II70, for the His-tagged SigB in E. coli. The over-expression and purification of SigB-His were performed the same as for PCC 6803 SigC-His as previously described (Imamura et al. 2003b).

Over-expression and purification of His-tagged RpoC1 and RpoC2

For in vitro transcription and the His-tag pull-down assay, N-terminal His-tagged RpoC1 and RpoC2 were purified. His-RpoC1 was overproduced and purified as previously described (Imamura et al. 2003b). For the over-expression of the rpoC2 gene, E. coli strain M15[pREP4] containing pQE1789C (Imamura et al. 2003b) was used. The over-expression and purification were performed the same as for PCC 6803 NtcA as reported previously (Asayama et al. 2004). The purified His-RpoC1 and His-RpoC2 obtained by Ni-NTA were subjected to SDS-PAGE and the proteins recovered from the gel were renaturated as previously described (Imamura et al. 2003b).

In vitro transcription

Multiple-round run-off assays were performed as previously described (Shibato et al. 1998) except that the transcription reaction was performed at 30 °C (referred to as standard conditions here). The heterologous and homologous RNAP holoenzymes comprised the E. coli core (Asayama et al. 1996) (1 pmol) or reconstituted PCC 6803 core enzyme (1 pmol) and each purified recombinant PCC 6803 sigma factor (Imamura et al. 2003b) (four-fold molar excess over the core enzyme). The constructs of template DNA used in this study are as follows: pKK-A2 (Imamura et al. 2003b); pKK-PA, a 518-bp segment of the BamHI-BglII PCC 6803 hspA promoter region (–467 to +51) derived from pHSPA (Imamura et al. 2003b), which was replaced with a 274-bp BamHI segment containing the tac promoter on pKK223-3; pKK-TA, a 438-bp segment of the PCR-amplified BamHI-BglII PCC 6803 lrtA promoter region (–497 to –59) cloned into pKK223-3 the same as pKK-PA; pKK-IY1 and pKK-IY4, a 104-bp segment of the BamHI K-81 psbA2 (–47 to +14) promoter region derived from pIY1 and pIY4 (Ito et al. 2003), respectively, cloned into pKK223-3 the same as pKK-PA. Each promoter region was inserted in the same orientation as the removed tac promoter, so an internal rrnBT1T2 terminator (Amann et al. 1983) was placed at downstream of the promoter regions. Transcripts synthesized in vitro by supercoiled PCC 6803 psbA2 (329 nt), PCC 6803 hspA (345 nt), PCC 6803 lrtA (502 nt), K-81 psbA2 (296 nt) and RNA I (107 nt) promoters were resolved by electrophoresis on a 5% or 4% polyacrylamide gel containing 8 M urea and then subjected to autoradiography.

His-tag pull-down assay

The purified His-RpoC1 (20 pmol) or His-RpoC2 (20 pmol), E. coli core enzyme (20 pmol), and/or bovine serum albumin (BSA) (40 pmol, 60 pmol or 100 pmol), or {alpha}-casein (40 pmol, 60 pmol or 100 pmol) were mixed. After incubation at 30 °C for 30 min, 100 µL of a 50% slurry of Ni-NTA equilibrated in TG20–20 buffer (20 mM Tris-HCl (pH 7.9), and 20% glycerol) containing 5 mM imidazole was added to the mixture and mixed gently for 30 min at room temperature. After centrifugation at 7000 g for 1 minute at room temperature to pellet the resin, the column was washed with 500 µL of TG20–20 buffer containing 5 mM imidazole and 500 µL of TG20–20 buffer containing 30 mM imidazole (5 times) and then the protein bound to the column was eluted with 100 µL of TG20–20 buffer containing 250 mM imidazole. The eluted fraction (15 µL) was resolved by 10% SDS-PAGE and subjected to Western blotting as previously described with PCC 6803 RpoC1, RpoC2, E. coli RpoA, RpoB or RpoC antibody (Imamura et al. 2003a, b).


   Supplementary material
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
References
 
The following material is available form: http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC808/GTC808sm.htmFigure S1 Phylogenetic analysis of RpoC1 and RpoC2 subunits. (A) Molecular structure of E. coli RpoC, PCC 6803 RpoC1 and RpoC2. Small boxes indicate highly conserved domains, A to H (Allison et al. 1985). (B) Phylogenetic trees for RpoC1 (left panel) and RpoC2 (right panel). Phylogenetic analysis was performed as previously described (Imamura et al. 2003b). Whole amino acid sequences of each subunit were used for alignments for photosynthesizing organisms. On the other hand, N- or C-terminal amino acid sequences (N/C) of non-photosynthesizing bacterial RpoC (B. subtilis, 1–612/613–1199; M. tuberculosis, 1–685/686–1316; E. coli, 1–595/596–1407; P. putida, 1–595/596–1399; and S. typhimurium, 1–595/596–1407), corresponding to the RpoC1/RpoC2 subunits of photosynthesizing organisms, were also used. The numbers at each node represent the percentage of trees supporting the specific branching pattern in the bootstrap analysis. The bar indicates the distances corresponding to 20 changes per 100 amino acid positions. Designations and GENBANK accession numbers for sequences of the subunits are as follows: B.subtilis-RpoC for Bacillus subtilis RpoC (NP_387989 [GenBank] ); M.tuberculosis-RpoC for Mycobacterium tuberculosis RpoC (P47769); E.coli-RpoC for Escherichia coli RpoC (P00577); P.putida-RpoC for Pseudomonas putida RpoC (P19176); S.typhimurium-RpoC for Salmonella typhimurium RpoC (NP_463023 [GenBank] ); PCC6803-RpoC1 and PCC6803-RpoC2 for Synechocystis sp. PCC 6803 RpoC1 and RpoC2 in CyanoBase, http://www.kazusa.or.jp/cyano/Synechocystis/;PCC7120-RpoC1 and PCC7120-RpoC2 for Anabaena sp. PCC 7120 RpoC1 and RpoC2 in CyanoBase, http://www.kazusa.or.jp/cyano/Anabaena/;BP1-RpoC1 and BP1-RpoC2 for Thermosynechococcus elongatus BP-1 RpoC1 and RpoC2 in CyanoBase, http://www.kazusa.or.jp/cyano/Thermo/;PCC7421-RpoC1 and PCC7421-RpoC2 for Gloeobacter violaceus PCC 7421 RpoC1 and RpoC2 in CyanoBase, http://www.kazusa.or.jp/cyano/Gloeobacter/;PCC7942-RpoC1 and PCC7942-RpoC2 for Synechococcus elongatus PCC 7942 RpoC1 and RpoC2 (P42079 and ZP_00164591); P.thunbergii-RpoC1 and P.thunbergii-RpoC2 for Pinus thunbergii RpoC1 and RpoC2 (P52733 and P41606); O.sativa-RpoC1 and O.sativa-RpoC2 for Oryza sativa RpoC1 and RpoC2 (P12092 and P12093); A.thaliana-RpoC1 and A.thaliana-RpoC2 for Arabidopsis thaliana RpoC1 and RpoC2 (P56763 and P56764); N.tabacum-RpoC1 and N.tabacum-RpoC2 for Nicotiana tabacum RpoC1 and RpoC2 (NP_054487 [GenBank] and NP_054486 [GenBank] ).


   Acknowledgements
 
We wish to thank Dr Akira Ishihama for technical advice and the gift of the E. coli RNAP antibodies and Dr Shigeyuki Imamura for technical advice.


   Footnotes
 
Communicated by: Nobuo Shimamoto

* Correspondence: E-mail: asam{at}mx.ibaraki.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
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Received: 15 June 2004
Accepted: 21 September 2004




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