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J. Biol. Chem., Vol. 278, Issue 34, 31701-31708, August 22, 2003

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RNA Polymerase Subunit Requirements for Activation by the Enhancer-binding Protein Rhodobacter capsulatus NtrC^*

Cynthia L. Richard, Animesh Tandon, Nathaniel R. Sloan and Robert G. Kranz 

From the Department of Biology, Washington University, St. Louis, Missouri 63130

Received for publication, April 28, 2003 , and in revised form, May 22, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Rhodobacter capsulatus NtrC is an enhancer-binding protein that activates transcription of the R. capsulatus ⁷⁰ RNA polymerase, but does not activate the Escherichia coli ⁷⁰-RNA polymerase at the nifA1 promoter. We utilized R. capsulatus:E. coli hybrid RNA polymerases assembled in vitro to investigate the subunits required for protein-protein interaction with RcNtrC at the nifA1mut1 promoter. Assembly of core Rc' or hybrid RNA polymerases containing the Rc' subunits absolutely require the inclusion of an subunit, with the Ec subunit only partially promoting RNA polymerase assembly. The RcEc' RNA polymerase is not activated by RcNtrC. Moreover, a mutant form of the Rc lacking the C-terminal domain, when assembled with the Rc' and ⁷⁰ subunits, is activated by RcNtrC. These results suggest that the R. capsulatus subunit is not important for RcNtrC interaction. All hybrid RNA polymerases that contained the Rc' were activated by RcNtrC, suggesting that the Rc' subunit plays an important role. It is proposed that RcNtrC recruits R. capsulatus ⁷⁰-RNA polymerase to the promoter through interaction with Rc'. RcNtrC interacts with RNA polymerase from a unique position, with dimers centered at –118 bp from the start site. Placing the RcNtrC tandem binding sites on the opposite face of the helix (–113 bp) completely abolished transcription activation. Moving the RcNtrC tandem binding sites 20 bp closer to or further from the promoter significantly reduced activation, again suggesting unique spatial constraints on how RcNtrC interacts with the R. capsulatus RNA polymerase.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In Rhodobacter capsulatus, a high GC photosynthetic -proteobacterium, the expression of many nitrogen fixation and nitrogen assimilatory genes is under the control of a two-component regulatory system comprised of RcNtrB and RcNtrC¹ (1). RcNtrB is a histidine kinase that autophosphorylates in response to nitrogen deprivation and the phosphate is subsequently transferred to the response regulator, RcNtrC (2). Phosphorylated RcNtrC activates transcription of promoters containing specific RcNtrC tandem binding sites situated >100 bp upstream of the transcription start site (3–6). RcNtrC shares some common features with Escherichia coli EcNtrC, such as the DNA binding position (i.e. where enhancer-binding proteins, EBPs, typically bind), an ATP requirement, and the nitrogen sensing and signaling mechanisms. However, differences from EcNtrC in the central domain (called region 3), the absence of ATP hydrolysis, and the fact that the RcNtrC activates the ⁷⁰ containing RNA polymerase (RNAP) rather than the ⁵⁴ containing RNAP, distinguishes RcNtrC from other EBPs in general (3, 5).

When genes are regulated by activator proteins, transcriptional activation requires direct contact between the activator protein and RNAP at the promoter (7, 8). RNAP is comprised of two , one each , ', , and subunits (9). A combination of genetics, cross-linking, and other biochemical approaches have demonstrated activator protein interactions with the , ⁷⁰, and more recently the and ' subunits (10, 11). Activator proteins that interact with ⁷⁰/RNAP typically interact with the (and/or ⁷⁰) subunits and thus represent the largest characterized types of activator-RNAP interactions. A well studied example is the E. coli catabolite activator protein (CAP) ⁷⁰-RNAP interaction (12). The interactions between CAP and the subunit of RNAP can be explained by whether CAP binds to DNA at –41.5 or –61.5 (class II and class I, respectively) (13, 14). Cross-linking evidence suggests that the Rhizobium meliloti EBP, DCTD activator, contacts ⁵⁴/RNAP via the and ⁵⁴ subunits (15, 16) and the bacteriophage N4 single stranded-binding protein activates the ⁷⁰/RNAP via the ' subunit (17). The N4 single stranded-binding protein mechanism is unique in that DNA binding is not required for activation (17). RcNtrC presents a unique system to study ⁷⁰/RNAP-EBP interactions because of the differences already noted above for this activator.

Protein cross-linking and genetic techniques have been the standard to study protein-protein interactions. However, previous proposals to use heterologous activators (from Pseudomonas spp.) and E. coli RNAPs have suggested another approach to studying such interactions (18). Hybrid RNAPs have been assembled between E. coli and, for example, Bordetella pertussis (19), and are beginning to prove useful in determining RNAP-activator protein interaction. The premise for this approach is that over hundreds of millions of years of evolutionary divergence, specific activator-RNAP interactions may coevolve. Thus determining which subunit(s) of RNAP from the natural bacterium are necessary for activation would suggest specific sites of interaction on that subunit with the activator. The proteobacteria and E. coli ( proteobacteria) are excellent candidates to test this approach, because they diverged more than 500 million years ago. While our study was in progress another study demonstrated that the Agrobacterium tumifaciens subunit is required for virulence gene activation by VirG both in vivo and in vitro using a hybrid RNAP with the A. tumifaciens subunit and E. coli , ', and ⁷⁰ subunits (20, 21). To date the only in vitro assembled hybrid RNAPs have been with a heterologous subunit and E. coli , ', and ⁷⁰ subunits (e.g. A. tumifaciens and Sinorhizobium meliloti) (20, 22). We have successfully assembled completely recombinant R. capsulatus RNAP (Rc'⁷⁰) and demonstrated that its in vitro activation by RcNtrC is similar to the natural RcRNAP (5).² Based on the availability of both R. capsulatus and E. coli recombinant purified RNAP subunits and the ability to assemble functional RNAPs in vitro, we have used the hybrid RNAP to study ⁷⁰-RNAP interactions with RcNtrC. In this study, we report the successful assembly of hybrid R. capsulatus/E. coli RNAPs and the determination that the Rc' subunit is necessary and sufficient for RcNtrC activation. We demonstrate that an subunit is essential for in vitro assembly when the Rc and Rc' subunits are components of the reconstitution. Finally, we show that the tandem RcNtrC binding sites have strict spatial and face of the helix constraints relative to the RNAP binding site (promoter) for transcription activation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Growth Media and Strains—E. coli strains were grown in Luria broth at 37 °C and R. capsulatus was grown in the minimal medium RB (23). R. capsulatus SB1003 is a spontaneous rifampicin-resistant wild type (24). E. coli strains TB1 and K-12 were common laboratory strains. E. coli strains BL21(DE3)pLysS and Tuner(DE3)pLacI were purchased from Novagen. Carbenicillin (Sigma), when used, was at a concentration of 150 µg/ml. Rifampicin (Sigma), when used in in vitro transcription reactions, was at a final concentration of 62.5 µg/ml.

Expression Plasmids—The E. coli RNAP subunit expression plasmids pHTT7f1-NH (25), pMKSe2 (26), pT7' (27), and pRGK329 have been described.² The R. capsulatus RNAP subunit expression plasmids pRGK301 (5), pRGK325, pRGK326, pRGK327, and pRGK328 have been described.² pRGK330, which allows overexpression of the E. coli subunit was made by PCR of the E. coli rpoZ gene from K-12 chromosomal DNA with the upstream oligonucleotide 5'-CGCCATGGCACGCGTAACTGTTCAGG-3' and the downstream oligonucleotide 5'-GCTCGAGACGACGACCTTCAGCAAT-3'. The 275-bp PCR product was digested with NcoI/XhoI and ligated in-frame to the carboxyl-terminal His₆ tag encoded by pETBlue2 (Novagen). pRGK331, which allows overexpression of the 257-amino acid NH₂-terminal domain of Rc (linker and COOH-terminal domain deletion mutant; Rc²⁵⁷) was made by PCR from the R. capsulatus rpoA gene of SB1003 with the upstream oligonucleotide 5'-CGGGATCCTCATTTCTTCAGCAGCAGCGGGTT-3' and the downstream oligonucleotide 5'-CGGGATCCTCAGAACTGGTCTTCGAAGCGCTTGGCC-3'. The 771-bp PCR product was digested with NdeI/BamHI and ligated in-frame to the amino-terminal His₆ tag encoded by pET15b (Novagen). All expression plasmids were sequenced with vector-specific oligonucleotide primers to confirm the proper orientation of the cloned insert, the junction sequence, and the correct open reading frame sequence. The partial sequence of the insert (250–350 bp from each end) that was obtained was identical to the R. capsulatus chromosomal sequence.³

Transcription Templates—pA1M1 (WT) and pALB2 (no NtrC binding sites) have been described previously (5, 28). pA1M1(+10) was made by PCR of pA1M1 with the left oligonucleotide 5'-AAACTCGAGGACAGGCAAGTTCGGTTC-3' and the right oligonucleotide 5'-TTTCTCGAGTCCCGGACGCGTCGGAAG-3'. The 3.5-kb product was digested with XhoI and recircularized. pA1M1(+20) was made by PCR of pA1M1 with the left oligonucleotide 5'-AAAAGATCTTGGACAGGCAAGTTCGGTTCA-3' and the right oligonucleotide 5'-TTTAGATCTGACACGAGTCCCGGACGCGTC-3'. The 3.5-kb product was digested with BglII and recircularized. pA1M1(–10) was made by PCR of pA1M1 with the left oligonucleotide 5'-AAACTCGAGGACAGGCAAGTTCGGTTC-3'. The 3.5-kb product was digested with XhoI and recircularized. pA1M1(–20) was made by PCR of pA1M1 with the left oligonucleotide 5'-TTCTCGAGTCACCGGGTCGAT-3' and the right oligonucleotide 5'-AACTCGAGTGCCGGACGCGTC-3'. The 3.5-kb product was digested with XhoI and recircularized. pA1M1(–5) was made by PCR of pA1M1 with the left oligonucleotide 5'-AACTCGAGTTCATTTTTGCAA-3' and the right oligonucleotide 5'-AACTCGAGTGCCGGACGCGTC-3'. The 3.5-kb product was digested with XhoI and recircularized. pA1M1(–24) was made by PCR of pA1M1 with the left oligonucleotide 5'-AACTCGAGCGGGTCGATTGCG-3' and the right oligonucleotide 5'-AGCTCGAGTGCCGGACGCGTC-3'. The 3.5-kb product was digested with XhoI and recircularized.

Purification of Rc, Ec, Crude , and ' Subunits, Rc⁷⁰, Ec⁷⁰, MBP-NtrB, RcNtrC, and Native RcRNAP—Purification of the His₆-tagged Rc and Ec subunits, Rc, ', Ec, ', and His₆-tagged Rc⁷⁰ and Ec⁷⁰ have been described previously (5).² Purification of the MBP-NtrB and RcNtrC has been described previously (2). R. capsulatus natural RNAP was isolated and purified as previously described by Cullen et al. (28). The native EcRNAP core and holoenzymes were purchased from Epicenter.

Rc²⁵⁷ Subunit Purification—The Rc²⁵⁷ subunit was purified based on a modification of the procedure of Tang et al. (25). The His₆ amino-terminal-tagged Rc²⁵⁷ subunit from R. capsulatus was overexpressed in E. coli strain BL21(DE3) containing pRGK331 by the addition of 1 mM isopropyl-1-thio--D-galactopyranoside for 3 h at 37 °C. Cells were harvested at 3000 x g and sonicated in 20 ml of buffer A (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole). The lysate was cleared by centrifugation at 16,000 x g for 20 min at 4 °C in a Sorvall centrifuge. By SDS-PAGE analysis, the induced supernatant contained a major polypeptide of 32 kDa that was not present in the uninduced sample. The volume of the supernatant was adjusted to 50 ml with buffer A and the Rc²⁵⁷ subunit was precipitated by the addition of (NH₄)₂SO₄ to 60%. The Rc²⁵⁷ subunit was collected by centrifugation at 16,000 x g for 20 min at 4 °C and was dissolved in 20 ml of buffer B (6 M urea, 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole). The sample was loaded onto a His-Bind (Novagen) column that had been washed after Ni²⁺ charging with buffer B and the column was washed with 10 column volumes of buffer B, followed by 6 column volumes of buffer C (6 M urea, 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 30 mM imidazole). The His₆-tagged Rc²⁵⁷ subunit of RNAP was eluted with 6 column volumes of buffer D (6 M urea, 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 500 mM imidazole) and 1-ml fractions were collected. The protein concentrations were determined with the Coomassie Plus-200 protein assay reagent (Pierce). The protein fractions were stored at –80 °C and were found to be stable for reconstitutions for up to 3 months.

Ec Subunit Purification—Carboxyl-terminal His₆-tagged -subunit from E. coli was overexpressed in E. coli strain Tuner(DE3)pLacI containing pRGK330. The culture was grown to an A₆₀₀ of 0.6–1.0 and protein expression was induced by the addition of 1 mM isopropyl-1-thio--D-galactopyranoside for 3 h at 37 °C. The cells were harvested at 3,000 x g for 15 min at 4 °C and sonicated in 20 ml of binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The supernatant was cleared by centrifugation at 12,000 x g for 20 min at 4 °C in a Sorvall centrifuge. The supernatant contained a major polypeptide of 10 kDa that was not present in the uninduced sample. The cleared supernatant was loaded onto a His-Bind column and the column was washed with 10 column volumes of binding buffer, followed by 6 column volumes of wash buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The His₆-tagged Ec subunit was eluted in 6 column volumes of elution buffer (250 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and 1-ml fractions were collected. The protein concentration of each fraction was determined (as above) and the fractions were stored at –80 °C for up to 3 months with no detectable loss of ability to efficiently reconstitute.

Reconstitution of RNAP Hybrid Enzymes—All hybrid RNAPs were reconstituted and purified as for the recombinant reconstituted R. capsulatus holoenzyme (Rc'⁷⁰).² The amount of RNAP subunits added per 2-ml reconstitutions are as follows: 60 µg of His₆-tagged , 300 µg of crude , 600 µg of crude ', 60 µg of His₆-tagged , and 120 µg of His₆-tagged Rc⁷⁰ or Ec⁷⁰. Combinations of R. capsulatus and E. coli RNAP subunits were combined in Snakeskin dialysis tubing (Pierce), the final reconstitution volume was brought to 2 ml. RNAPs prepared in this way were stored at –20 °C and were found to be stable for at least 3 months.

In Vitro Transcription—In vitro transcription assays were performed as described below, according to Bowman and Kranz (5) in a 16-µl reaction containing the transcription buffer (50 mM Tris-HCl, pH 8, 100 mM potassium acetate, 10 mM magnesium acetate, 1 mM ATP, 10 mM dithiothreitol, and 0.5 µl of RNasin (Promega)). The concentration of nifA1mut1 supercoiled template was 40 nM. For transcriptional activation 270 nM MBP-NtrB was incubated at 37 °C for 10 min in transcription buffer before the simultaneous addition of 320 nM NtrC and 40 nM RNAP. The reactions were incubated for 10 min at 24 °C and start solution (50 µg/ml heparin; 0.4 mM ATP, GTP, and UTP; 0.01 mM CTP; 0.5 µl of 25 µCi of [-³²P]CTP (PerkinElmer Life Sciences)) was added and incubation continued at 24 °C for 30 min. The reactions were terminated by the addition of 7 µl of stop solution (95% formamide, 1 mM EDTA, 0.025% xylene cyanol, 0.025% bromphenol blue), heated to 70 °C for 3 min, and loaded onto an 8% acrylamide sequencing gel. Purified ⁷⁰ proteins (tagged with His₆) were added to the core RNAPs prior to the start of the reactions.

Other Methods—Transcripts were quantitated by digitizing the autoradiograms with a Fuji Luminescent Image Analyzer (LAS-1000 plus) and analyzing the bands by use of Fuji Image Gauge software (version 3.4). Both the digitizing and software could readily distinguish differences in transcript levels of 2-fold or more. The quantitation values indicated in parentheses are an average of the percent of nifA1mut1/RNAI for a minimum of three in vitro transcription experiments. The background was subtracted for each lane independently.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Subunit-RcNtrC Interaction Requirements—To investigate which subunits are involved in RcNtrC-mediated transcription activation, we have combined the use of the nifA1mut1 promoter (Fig. 1A) and the ability to assemble hybrid RNAPs with R. capsulatus and E. coli subunits (see below). We have demonstrated that the R. capsulatus RNAP (Rc'⁷⁰) can be assembled from purified recombinant subunits by a modified method of Tang et al. (25) and that this enzyme has the same RcNtrC activation properties as the RNAP isolated from R. capsulatus.² We chose the nifA1mut1 promoter (Fig. 1A) because of the strong RcNtrC dependence for activated transcription (e.g. compare Fig. 1, C, lane 2 with lane 4) and the extensive studies already completed on the nifA1 promoter in vivo and in vitro (5). Moreover, the native ⁷⁰ RNAP from E. coli is not activated by RcNtrC at this promoter (3, 5). All of the hybrid enzymes assembled for the present study are denoted by the source organism, either R. capsulatus (Rc) or E. coli (Ec), immediately prior to the specific subunit(s) designation. All hybrid RNAPs were >95% pure as determined by SDS-PAGE (e.g. Fig. 1B, lane 1).

View larger version (43K): [in this window] [in a new window]   FIG. 1. DNA sequence of the nifA1 and nifA1mut1 promoters, SDS-PAGE of RcEc', and in vitro transcription reactions at the nifA1mut1 promoter. A, nucleotide sequence of the nifA1 and nifA1mut1 promoters. The transcription start site is designated +1 and the –10 and –35 sequences are underlined. Shaded bars indicate the RcNtrC tandem binding sites. B, 12.5% SDS-PAGE of RcEc' hybrid RNAP with 25 µg of protein sample/lane. Molecular size standards (Bio-Rad Low Range) are shown on the right, RNAP subunit(s) location are shown on the left, and lane numbers are given at the bottom. Lane 1, assembled RcEc' hybrid RNAP; lane 2, low molecular weight size standard. C, in vitro transcription assays on the nifA1mut1 template. The positions of the RNAI (108 nucleotides) and nifA1mut1 (97 nucleotides) transcripts are shown on the right and the RNAP is noted above. The percentage of RNAI of the nifA1mut1 transcript is given in parentheses below the lane numbers (e.g. 100% would indicate that RNAI and nifA1mut1 transcripts are equal).

Because the RcNtrC system activates the ⁷⁰ RcRNAP and all well studied bacterial activators of this RNAP interact with the subunit (or ⁷⁰ or both) (29), we chose to initially reconstitute a hybrid enzyme with all E. coli subunits and the R. capsulatus . Hybrid RNAPs were assembled with the amino-terminal His₆-tagged Rc subunit and the E. coli and ' subunits. As shown in Fig. 1B, RcHis₆ (40 kDa) was able to successfully assemble with the Ec and Ec' (150 kDa) subunits as for RNAP core enzyme. RcEc' is highly active at the vector RNAI promoter when either Rc⁷⁰ or Ec⁷⁰ is added (Fig. 1C, lanes 14 and 15). The Ec subunit was originally cloned from a rifampicin-sensitive strain, whereas the Rc subunit in the present study was cloned from a rifampicin-resistant strain (24). Thus, hybrid enzymes can be probed for proper composition by the use of rifampicin to inhibit transcription. The addition of rifampicin completely abolished all activity of RcEc' indicating that the rifampicin-sensitive Ec subunit was properly assembled (not shown). The RcEc' RNAP has the same RcNtrC activation properties as the E. coli RNAP (Fig. 1C, lanes 9 and 10 versus lanes 16 and 17), suggesting that the Rc subunit is not sufficient to allow RcNtrC activation even when the Rc⁷⁰ is used for promoter recognition (Fig. 1C, lane 16). No significant amount of nifA1mut1 transcript could be detected with RcEc' upon addition of RcNtrB/NtrC (Fig. 1C, compare lanes 4 and 5 with lanes 16 and 17). As noted previously, the RcRNAP is activated by RcNtrC at this promoter (Fig. 1C, lanes 2 and 3 compared with lanes 4 and 5).

The R. capsulatus subunit is 45% identical to the E. coli subunit and the Rc COOH-terminal domain (-CTD) is separated from the Rc NH₂-terminal domain by a 17-amino acid linker. The -CTD has been shown to be required for many ⁷⁰ RNAP activators but is not required by the ⁵⁴ type activators (11, 12). In E. coli it is possible to delete the subunit CTD and maintain efficient in vitro assembly and activity of the RNAP (12). To determine whether the Rc-CTD is required for RcNtrC-mediated transcription activation, we removed the Rc-CTD (called Rc²⁵⁷) and assembled this with all recombinant R. capsulatus RNAP subunits (Rc²⁵⁷'⁷⁰). The Rc²⁵⁷ mutant is hexahistidine tagged on the amino terminus and contains the 257 amino acids comprising the -NH₂-terminal domain but does not include the flexible linker or the proposed Rc-CTD. In the presence of RcNtrB and RcNtrC an approximate 6-fold activation of the nifA1mut1 promoter is detected (Fig. 2A, lane 2), which is comparable with the activation seen with the intact Rc (Fig. 2B, lane 2). This result supports the observation that the Rc subunit is not required for RcNtrC-mediated activation.

View larger version (38K): [in this window] [in a new window]   FIG. 2. In vitro transcription of the nifA1mut1 promoter with assembled Rc257'70. The positions of the RNAI and nifA1mut1 transcripts are shown to the right, with the percentage of RNAI given below in parentheses.

Subunit—In E. coli the subunit of RNAP is not required for in vitro renaturation of enzyme activity and an -less strain is viable, exhibiting a slow growth phenotype (30). The subunit is evolutionarily conserved across bacteria, archaea, and eukaryotes (31). In bacteria each of the three conserved regions of interacts with a different region of the ' subunit and is proposed to function by preventing aggregation of ', thus aiding proper folding and assembly of ' onto the ₂ subassembly, under some conditions (31–33). Nevertheless, in vitro assembly of the E. coli RNAP (Ec') does not require subunit (Fig. 3, lanes 14–16) as shown previously (e.g. Ref. 34). Our initial studies of the Rc' assembly resulted in inactive RNAP, so we cloned and overproduced the Rc subunit to determine whether this would aid assembly. We have previously shown that a protein of 23 kDa co-purifies with the RcRNAP and that this protein is the R. capsulatus subunit (5). The Rc subunit is 39% identical to the E. coli subunit and contains the three conserved regions. The most notable difference between the R. capsulatus and E. coli subunits is the presence of an additional 22 amino acids on the COOH-terminal end of Rc. Surprisingly the reconstitution of active R. capsulatus core RNAP required the subunit. Whereas Rc' core RNAP could not be reconstituted into a functional RNAP (e.g. Fig. 3, lane 2, RNAI transcript), when Rc is included, transcription is observed (Fig. 3, lane 6, RNAI transcript). The addition of bovine serum albumin to the Rc' reconstitutions in amounts equivalent to or in excess to those of Rc did not yield any transcription activity, suggesting that the Rc subunit is specifically required.

View larger version (33K): [in this window] [in a new window]   FIG. 3. Recombinant subunit assembly comparisons. In vitro transcription reactions of the nifA1mut1 promoter and added activators and factors are noted. The assembled RNAP is noted above. The positions of the RNAI and nifA1mut1 transcripts are shown to the right and the percentage of RNAI, in parentheses, is given below.

To determine whether the E. coli subunit can replace the Rc subunit for assembly and RcNtrC activation, we cloned and overexpressed Ec. The addition of the Ec subunit to the Rc' core RNAP reconstitutions allowed assembly into an active RNAP but consistently only to a level that is 50% of the core enzyme reconstituted with the Rc (Fig. 3, RNAI transcript, lane 6 compared with lane 10). With our in vitro reconstitution and transcription system the Ec' core RNAP reconstitutes to approximately equivalent activity levels with either the Rc or Ec as without the addition of any subunit (Fig. 3, compare RNAI transcript of lanes 16, 20, and 24).

The subunit of RNAP, when covalently linked to a DNA-binding protein, can function as a transcription activator, presumably by its interaction with RNAP and ' (29). To determine whether the Rc subunit functioned specifically in protein-protein contacts with RcNtrC, we compared RcNtrC activation profiles at the nifA1mut1 promoter with RNAPs reconstituted with either Rc or Ec subunits. In these experiments, the ⁷⁰ subunit was added to reactions with core rather than inclusion in the assembly step. The R. capsulatus core RNAP (Rc') can be activated by RcNtrC on the nifA1mut1 template to a level that is comparable with that of reconstituted holoenzyme (Fig. 2B, lane 2, and Fig. 3, lane 8). When the Ec subunit replaces the Rc subunit for in vitro reconstitution of the Rc' core RNAP, RcNtrC-mediated activation is detected (Fig. 3, lane 12). However, the activation level consistently averages only 40% of the level seen with the Rc (Fig. 3, lane 8 versus lane 12). The E. coli core RNAP reconstituted with Rc is not activated by RcNtrC to a detectable level (Fig. 3, lane 20). These results suggest that the Rc subunit is not sufficient or essential for RcNtrC transcription activation (also see below).

Rc-RcNtrC and Rc'-RcNtrC Interaction—To determine whether the Rc and Rc' subunits are sufficient for RcNtrC activation we assembled hybrid RNAPs that contained the Ec, Rc, and Rc' subunits, either individually or in combination with other recombinant RcRNAP subunits. The assembly requirement for the subunit was further defined when Rc and Rc' are present in combination in the hybrid RNAP (Fig. 4, RNAI, lanes 4, 8, and 12). When either the Rc or Ec subunits are used to reconstitute EcRc', RcNtrC activation is detected (Fig. 4A, lanes 8 and 12). This result suggests that RcNtrC is interacting with the Rc and/or the Rc' subunit(s). When the only E. coli subunit present in the hybrid enzyme is Ec', no significant activation of nifA1mut1 is detected (Fig. 4B, lane 4). When only the Ec subunit is present in the hybrid enzyme, activation of nifA1mut1 is detected (Fig. 4C, lane 4), regardless of the ⁷⁰ used (Fig. 4C, lane 5). These results suggest that the Rc' is critical for activation by RcNtrC. To confirm this, we assembled a hybrid holoenzyme where the only core subunit from R. capsulatus is Rc'. With this hybrid RNAP, RcNtrC-mediated transcriptional activation is observed (Fig. 4D, lane 2). These results demonstrate that the Rc' subunit is sufficient to confer RcNtrC activation and that RcNtrC may interact with the Rc' subunit (see "Discussion").

View larger version (45K): [in this window] [in a new window]   FIG. 4. In vitro transcription of the nifA1mut1 promoter with specified combinations of RNAP recombinant subunits. The position of the RNAI and nifA1mut1 transcripts is shown on the right and additional components are noted. The hybrid RNAP is noted above. The percentage of RNAI of the nifA1mut1 transcript is given in parentheses below the relevant lane numbers.

The Upstream Distance Requirements for Activation by RcNtrC—One of the defining characteristics of EBPs of the NtrC class is the distance from the promoter where EBP dimers bind (16). RcNtrC is no exception to this and in the 6 natural promoters that are activated by RcNtrC, the tandem binding sites are centered from –102 to –133 bp upstream (3–6). In the case of the nifA1 promoter the activator binding sites are centered at –118 bp upstream of the transcription start site (Fig. 5A). Such a spatial requirement places constraints on where RcNtrC may interact with the RNAP. The suggestion that the Rc' subunit is sufficient for interaction may be a ramification of such spatial requirements (see Fig. 6 model). From similar logic, nearly all ⁷⁰/RNAP activators that interact with the (and/or ) subunits bind adjacent to the RNAP where face of the helix is important. We wanted to determine whether the RcNtrC tandem binding sites could be moved closer to the RNAP –35/–10 binding regions and whether the face of the helix presenting RcNtrC to RNAP is important. To determine the spatial limits for RcNtrC site placement, we constructed a defined set of insertions or deletions between the proximal RcNtrC binding site and the transcription start site of the nifA1mut1 promoter (Fig. 5, A and B). We have previously shown that both RcNtrC binding sites are required for activated transcription in vivo and in vitro (3, 5). Transcription is activated to near wild type levels when the RcNtrC tandem binding sites are moved 10 bp closer (–108) to the transcription start site (Fig. 5B). However, a 5-bp deletion shows only background levels of transcription, indicating a face of the helix requirement. The optimal level of RcNtrC transcription activation occurs between –128 and –108. Both the 20-bp insertion and 20-bp deletion significantly reduce the level of activation, consistent with the limits for the RcNtrC tandem binding sites observed with the naturally occurring promoters. These results are consistent with a model whereby spatial constraints exist on how RcNtrC contacts the RcRNAP (see Fig. 6 and "Discussion").

View larger version (18K): [in this window] [in a new window]   FIG. 5. DNA spatial requirement for RcNtrC activation. A, DNA sequence of the nifA1mut1 promoter. The transcription start site is shown with a horizontal arrow; the predicted –10 and –35 sites are boxed. The shaded bars represent the tandem RcNtrC binding sites. The sites of the deletions and insertions are noted. B, bar graph representing RcNtrC quantitated activation of the nifA1mut1 insertion and deletion templates. The respective number of inserted or deleted nucleotides is shown below each bar with the corresponding center of the RcNtrC tandem biding sites given below the insertion/deletion designation.

View larger version (32K): [in this window] [in a new window]   FIG. 6. Cartoon of RcRNAP-RcNtrC protein-protein interaction.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Our results with the EcRc'⁷⁰ hybrid RNAP represent the first instance of the ' subunit interacting with a bacterial DNA-binding activator protein. Currently, the only other example of an activator protein interacting with the ' subunit is with N4 single stranded-binding protein, which does not require DNA binding for activity (17). One caveat for the interpretation of our results is the assumption that "a requirement for activation and/or interaction" is related to a need for direct protein-protein contact. We cannot rule out a less direct interpretation, for example, recruitment occurs but the ' subunit is specifically necessary to mediate the transcription. Verification of direct protein-protein interaction will await cross-linking and/or genetic approaches.

When Rc' subunits alone were assembled, no RNAP activity could be detected. This suggested a requirement for an additional factor, most likely the subunit, as it is highly conserved and present in the natural RcRNAP. Unlike the in vitro assembly of core EcRNAP, in vitro assembly of the core RcRNAP absolutely requires the addition of an subunit. Utilizing standard genetic techniques, we have been unable to construct an -less (RpoZ^–) R. capsulatus strain,⁴ suggesting a specific requirement for for in vivo assembly of RNAP.

The subunit of RNAP has been shown to play an essential role in the transcription of many operons in E. coli and in other bacterial systems such as B. pertussis (19), Vibrio fischerii (35), Bacillus subtilis (36), Rhodospirillum rubrum (37), S. meliloti (22), and A. tumifaciens (20). The current model for activators that interact with the -CTD indicate two requirements: the activator must bind DNA upstream of the promoter and favorable protein-protein interactions must be made. In E. coli, a fully extended flexible linker allows the -CTD to extend 44 Å (12) thus permitting Ec⁷⁰ RNAP -CTD to interact with the TyrR EBP at the tyrP and mtr promoters (TyrR boxes centered at –64.5 and –77.5, respectively) (38–40). Studies with a synthetic CAP-activated promoter have shown that the CAP binding sites can be moved up to 91 bp from the transcription start site and CAP activation is maintained, albeit at a significantly reduced level (9% of wt) (41). We have shown that the -CTD is not required for RcNtrC activation at the nifA1mut1 promoter. Because the RcEc' RNAP is highly active at the RNAI promoter, the most likely explanation for our results is that the proper protein-protein interactions with RcNtrC are not met by the Rc. These results further distinguish RcNtrC ⁷⁰ RNAP activation from TyrR ⁷⁰ RNAP activation and other ⁷⁰ activators.

Our results suggest a model for RcNtrC activation (Fig. 6) in which the DNA between the RcNtrC tandem binding sites and the promoter loops out (or wraps around RNAP) allowing RcNtrC to contact the Rc' subunit. The significant decrease seen in RcNtrC activation with the 20-bp deletion supports spatial constraints for contacting the Rc'. Because we have been unable to demonstrate IHF binding or potential binding sites (3) and the fact that we see RcNtrC activation with our in vitro assembled Rc'⁷⁰, which is >95% pure, suggests that this looping (or wrapping) of the intervening DNA is naturally occurring. Based on our results we propose the mechanism for RcNtrC activation is recruitment, whereby RcNtrC oligomerizes at its tandem binding sites and recruits the ⁷⁰ RNAP to the promoter via an interaction with the ' subunit.

	FOOTNOTES

 
^* This work was supported by United States Department of Agriculture NRI Grant 99-35305-8647 (to R. G. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 314-935-4278; Fax: 314-935-4432; E-mail: kranz{at}biology.wustl.edu.

¹ The abbreviations used are: RcNTrC, Rhodobacter capsulatus nitrogen regulatory protein C; Ec, Escherichia coli; NtrC, nitrogen regulatory protein C; RNAP, RNA polymerase; EBP, enhancer binding protein; ⁷⁰, -70 factor; ⁵⁴, -54 factor; , subunit of RNAP; , subunit of RNAP; ', ' subunit of RNAP; , subunit of RNAP; CAP, catabolite activator protein.

² C. L. Richard, A. Tandon, and R. G. Kranz, manuscript in preparation.

³ wit.mcs.anl.gov/WIT2.

⁴ C. L. Richard and R. G. Kranz, unpublished results.

	ACKNOWLEDGMENTS

 
We thank Bill Bowman for some in vitro transcription templates.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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