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Nucleic Acids Research Pages 5630-5635  

The C-terminal carboxy group of T7 RNA polymerase ensures efficient magnesium ion-dependent catalysis
Introduction
Materials And Methods
   Preparation of T7 RNA polymerase and intein-containing precursor
   Preparation of C-terminal carboxy group-modified T7 RNA polymerase
   Elongation rate experiments
Results
   Introducing modifications of the C-terminal carboxy group of T7 RNA polymerase
   Development of an elongation rate assay for T7 RNA polymerase
   T7 RNA polymerase modified at the C-terminal carboxy group exhibits a magnesium ion-dependent decrease in the elongation rate
Discussion
Acknowledgements
References

The C-terminal carboxy group of T7 RNA polymerase ensures efficient magnesium ion-dependent catalysis

The C-terminal carboxy group of T7 RNA polymerase ensures efficient magnesium ion-dependent catalysis

Jens Lykke-Andersen and Jan Christiansen*

RNA Regulation Centre, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83 H,DK-1307 Copenhagen K, Denmark

Received August 31, 1998; Revised and Accepted October 28, 1998

ABSTRACT

DNA and RNA polymerases use divalent metal ions for catalysis. Crystal structures of several polymerases reveal that two acidic residues are involved in coordinating two metal ions at the catalytic centre. Bacteriophage RNA polymerases contain a highly conserved C-terminus with the carboxylate positioned near the active site. We examined whether theC-terminal carboxy group of T7 RNA polymerase is important for magnesium ion-dependent catalysis. Introduction of a methyl ester or decarboxylation of the C-terminal carboxy group was achieved with an intein-based protein expression system and an elongation rate assay was developed to test the effects of the modifications. The results show that enzymes with a modified C-terminal carboxy group exhibit a magnesium ion-dependent decrease in catalytic activity.

INTRODUCTION

Crystal structures of six DNA polymerases (1-9), two reverse transcriptases (10-12) and a DNA- and an RNA-dependent RNA polymerase (13,14) have provided evidence for a unified two metal ion catalytic mechanism of polymerases (reviewed in 15). This mechanism was first suggested from the crystal structure of the Klenow fragment of DNA polymerase I from Escherichia coli (16) and the hypothesis was later strongly supported by the structures of the ternary complexes of rat DNA polymerase [beta], T7 DNA polymerase and Bacillus stearothermophilus DNA polymerase I with substrates and metal ions (2,3,17). Two invariant acidic amino acid residues are positioned at the catalytic centre of all polymerase structures. Mutations of these invariant residues lead to strongly impaired polymerase activity (18-24) and the ternary complex crystal structures show that they are involved in coordinating the two catalytic metal ions (2,3,17). Moreover, a third acidic side chain is found at the active site in all polymerase structures except in T7 RNA polymerase, but this carboxylate is not superimposable (9).

T7 RNA polymerase is the only DNA-dependent RNA polymerase that has been crystallized (13,25). The structure resembles a right hand with the active site positioned in the palm. As in other polymerase structures two acidic residues (Asp537 and Asp812) are positioned near the catalytic centre and mutational studies have shown that they are important for catalysis (21-24). Moreover, electron paramagnetic resonance experiments on mutated T7 RNA polymerase have demonstrated that they are involved in metal ion binding (26). Bacteriophage RNA polymerases contain a highly conserved hydrophobic C-terminal amino acid sequence (27) that is poorly ordered in the electron density maps (13,25) and flexible in solution (27). However, the C-terminus of T7 RNA polymerase is positioned close to the active site (Fig. 1; 13) and mutational analyses have shown that both the length and the sequence of the C-terminal -Phe-Ala-Phe-Ala-OH883 `foot' is important for polymerase activity (27,28).

In this study we examined whether the C-terminal carboxy group is important for magnesium ion-dependent catalysis by T7 RNA polymerase. A recently developed intein-based protein purification system was implemented to generate T7 RNA polymerase derivatives with modified C-termini. The modified enzymes exhibited a magnesium ion-dependent decrease in catalytic activity, implying that the C-terminal carboxy group is important for magnesium ion-dependent catalysis.


Figure 1. Structure of the palm subdomain of T7 RNA polymerase. The positions of the C[alpha] atoms of the two active site acidic residues, Asp537 and Asp812, and the C-terminal carboxylate investigated in this study (Ala-OH883), are indicated. The figure was generated using coordinates from the published T7 RNA polymerase structure (13) and employing the programs MOLSCRIPT v.2.0 (33) and render from the Raster3D package (34).

MATERIALS AND METHODS

Preparation of T7 RNA polymerase and intein-containing precursor

Unmodified T7 RNA polymerase and a Cys883 mutant were expressed in E.coli as fusion proteins with glutathione S-transferase (GST) from pGEX-2TK (Pharmacia) derivatives and purified as described previously (29). The T7 RNA polymerase precursor used for modification of the C-terminal carboxy group was expressed in E.coli using a pCYB1 (New England Biolabs) derivative, pCYB1-T7RP-[Delta]Ala883, containing the T7 RNA polymerase open reading frame (ORF), without the C-terminal alanine codon ([Delta]Ala883), in-frame with the ORFs of the intein from the Saccharomyces cerevisiae VMA1 gene and a chitin binding domain (CBD). Escherichia coli DH5[alpha] containing the pCYB1-T7RP-[Delta]Ala883 plasmid was grown to an A600 of 0.8 in 1 l of LB medium containing 100 µg/ml ampicillin, before induction with 0.5 mM isopropyl [beta]-d-thiogalactopyranoside at 30°C for 6 h. Harvested cells were lysed by sonication in 40 ml 10 mM Tris-HCl (pH 8.0), 50 mM KCl (T10K50) containing 0.2 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin and 2 µg/ml aprotinin (Sigma). Triton X-100 was added to 0.5% and cell debris was removed by centrifugation. A 350 µl chitin suspension (New England Biolabs) was washed twice with 1 ml T10K50 and added to the cell lysate followed by incubation at room temperature for 30 min with gentle shaking. The main part of the supernatant was removed from chitin by centrifuging at 1500 g for 5 min and the chitin suspension was transferred to a 2 ml Eppendorf tube and washed five times with 1.5 ml T10K50 with intermittent centrifugations at 1500 g for 2 min. The chitin was resuspended in a total of 2 ml T10K50 containing 50% glycerol and 0.1% Triton X-100 and stored in 180 µl aliquots at -80°C.

Preparation of C-terminal carboxy group-modified T7 RNA polymerase

In order to modify the C-terminus of T7 RNA polymerase one tube of chitin containing T7 RNA polymerase [[Delta]Ala883]-intein CBD fusion protein, prepared as described above, was washed with 1 ml T10K50. The chitin was subsequently washed with 50 µl 50 mM Tris-HCl (pH 7.0), 0.1% Triton X-100 containing 50 mM l-cysteine, l-cysteine methyl ester or 2-mercaptoethylamine (cysteamine) and resuspended in the same solution and incubated at 4°C for 16 h. The supernatant was transferred to a new tube, glycerol was added to 35% and the protein stored at -20°C.

Elongation rate experiments

Elongation rates were measured using a HindIII-linearized pUC19 derivative, pUT7-L11-RNA1, containing the T7 [phiv]10 promoter and giving rise to an in vitro transcript of 2777 nt (30). Initially, T7 RNA polymerase was stalled at nt 26-28 by incubating 10 nM HindIII-linearized pUT7-L11-RNA1 plasmid with ~5 nM T7 RNA polymerase in 40 mM Tris-HCl (pH 8.0), 5 mM DTT, 1 mM spermidine, 0.1 mM ATP, GTP and UTP and, depending on the experiment, 0.5-5 mM MgCl2 at 22°C for 2 min. [[alpha]-32P]CTP (3000 Ci/mmol; Amersham) was added at 10 nM and incubation continued for 1 min before `re-booting' elongation with 0.5 mM of each nucleoside triphosphate and 50 mM KCl (we find that the addition of K+ after the polymerase stalling step is important for reproducible results). Elongations were stopped by 1 vol ice-cold formamide/10 mM EDTA at different time points. In experiments with time points at each second, the reaction was carried out in the tip of an automatic pipette (Eppendorf) and 5 µl samples were ejected each second. In mixing experiments, where two different polymerases were tested in the same tube, polymerases were stalled on their DNA templates in separate tubes, before they were combined and elongation continued in the presence of the four nucleoside triphosphates. Transcripts were denatured at 95°C for 30 s before separation in 5% polyacrylamide-7 M urea gels. Gels were subsequently subjected to autoradiography.

RESULTS

Introducing modifications of the C-terminal carboxy group of T7 RNA polymerase

The C-terminal carboxy group of T7 RNA polymerase was modified using a recently developed intein-based protein purification system (Materials and Methods). T7 RNA polymerase, lacking the C-terminal alanine ([Delta]Ala883), was expressed as a fusion with a chitin binding domain and the yeast VMA intein, which is mutated so that it can only participate in the first step of protein splicing (Fig. 2A; Materials and Methods). After purification on chitin, the thioester bond between Phe882 of T7 RNA polymerase and Cys1 of the intein, which is generated in the first step of protein splicing (31), can be cleaved by a nucleophile such as a thiol group (Fig. 2A). When cysteine is used as the nucleophile an S-N acyl rearrangement will take place after the initial nucleophilic attack, generating a normal peptide bond between the C-terminal cysteine and the rest of the enzyme (Fig. 2A; 31). This results in a full-length T7 RNA polymerase protein with a C-terminal Ala883->Cys883 mutation. By replacing the cysteine nucleophile with either cysteine methyl ester or 2-mercaptoethylamine, we were able to introduce modifications of the C-terminal carboxy group of T7 RNA polymerase (Fig. 2B).

Development of an elongation rate assay for T7 RNA polymerase

In order to generate an elongation rate assay for T7 RNA polymerase that circumvents the rate-limiting initiation step, we used a 2815 bp plasmid, pUT7-L11-RNA1. The plasmid contains the T7 [phiv]10 promoter, followed by a 14 bp sequence without cytidines in the non-template strand (Fig. 3A). In this way, it is possible to stall the elongating T7 RNA polymerase at positions 27-29 on the template, with an RNA labelled to high specific activity, by incubating the polymerase with ATP, GTP, UTP and low amounts of [[alpha]-32P]CTP (Fig. 3). Elongation is continued by adding all four unlabelled nucleoside triphosphates and samples are stopped at different time points and analysed by denaturing polyacrylamide gel electrophoresis (Fig. 3B). In contrast to a procedure that omits the elongation stalling step, the present approach exhibits reasonably homogeneous transcript lengths at the various time points and circumvents the rate-limiting initiation step which lasted >3 s at 22°C.

Table 1. Relative elongation ratesa of T7 RNA polymerase C-terminal derivatives at 22°C
T7 RNAP 5 mM Mg2+ 2 mM Mg2+ 1 mM Mg2+ 0.5 mM Mg2+
Wild-type 1.12 ± 0.03 (3) nd 1.14 ± 0.05 (3) nd
[Cys883] 1.00 (3) 1.00 (2) 1.00 (3) 1.00 (2)
[Cys-OMe883] 0.97 ± 0.04 (3) 0.52 ± 0.06 (2) 0.40 ± 0.05 (3) 0.30 ± 0.04 (2)
[decarboxy-Cys883] 1.01 ± 0.04 (3) 0.84 ± 0.10 (2) 0.62 ± 0.04 (3) 0.53 ± 0.07 (2)
aElongation rates relative to that of the T7 RNA polymerase [Cys883] mutant, determined from two to three independent experiments as shown in parentheses. Standard deviations are indicated. nd, not determined. A ratio of 1.00 corresponds to elongation rates of 79, 55, 9.8 and 3.0 nt/s at 5, 2, 1 and 0.5 mM Mg2+, respectively.

In order to modify the C-terminal carboxy group of T7 RNA polymerase an Ala883->Cys883 mutation had to be introduced (above), so the elongation rates of the unmodified T7 RNA polymerase and the Cys883 mutant were compared at 5 and 1 mM Mg2+ (Fig. 4). The polymerases were expressed as a fusion protein with an N-terminal GST tag which, after purification, was removed by thrombin. This leaves an N-terminal extension of nine amino acids, which have no effect on the elongation rate (data not shown). The polymerase was stalled at positions 27-29 at 5 or 1 mM magnesium ions and, after addition of 0.5 mM NTPs, samples were stopped every second (5 mM Mg2+) or every 10 s (1 mM Mg2+). At 5 mM Mg2+, the elongation rate of the unmodified enzyme was calculated as 88 nt/s at 22°C, whereas the rate for the Cys883 mutant was 79 nt/s (Fig. 4C and Table 1). This is two to three times slower than the reported elongation rate at 37°C (23). At 1 mM Mg2+, the elongation rate was ~8-fold slower than at 5 mM, with values of 11.2 and 9.8 nt/s for the wild-type and the Cys883 mutant, respectively (Fig. 4C and Table 1). In conclusion, the Cys883 mutant exhibited an elongation rate of 88-90% of the unmodified polymerase at both high and low magnesium ion concentrations. Extrapolating the transcript lengths to the 0 time point shows that a lag period does not occur after the stalled polymerase has been re-started with all four nucleoside triphosphates (Fig. 4C).


Figure 2. Preparation of C-terminal carboxy group-modified T7 RNA polymerase. (A) Preparation of the T7 RNA polymerase Cys883 mutant. T7 RNA polymerase lacking the C-terminal Ala883 ([Delta]Ala883) was expressed as a fusion with yeast VMA intein and a chitin binding domain (CBD) (Materials and Methods). An N-S acyl rearrangement occurs at Cys1 of the intein and the resulting thioester bond is susceptible to nucleophilic attack by the thiol group of an external cysteine (31). This generates a T7 RNA polymerase with a thioester-linked cysteine at the C-terminus (S-Cys883), which undergoes an S-N acyl rearrangement that generates T7 RNA polymerase with a C-terminal cysteine (Cys883). The acyl group of Phe882 is also shown in the drawings. (B) C-termini of the four T7 RNA polymerase derivatives used in the present study. The Cys883 mutant, generated as shown in (A), contains an extra thiol group compared with the unmodified polymerase (T7 RNAP). The Cys-OMe883 derivative was generated as in (A), by replacing l-cysteine with l-cysteine methyl ester as the external nucleophile. The decarboxy-Cys883 derivative was generated by replacing l-cysteine with 2-mercaptoethylamine.


Figure 3. T7 RNA polymerase elongation rate assay. (A) Outline of the linearized pUT7-L11-RNA1 plasmid used in the elongation rate assay. The T7 [phiv]10 promoter is shown as a black box and the sequence of the initial 32 nt of the resulting RNA transcript is shown with cytidines in bold upper case lettering. (B) Elongation rate assays of T7 RNA polymerase using the pUT7-L11-RNA1 plasmid. At the indicated Mg2+ concentration, the polymerase was stalled with a 26-28 nt transcript labelled to high specific activity (0 s, Materials and Methods) and transcription was continued in the presence of all four nucleoside triphosphates. Elongation was stopped after the time indicated above each lane. The resulting transcripts were separated in a high and low percentage denaturing polyacrylamide gel (upper and lower panels, respectively) and visualized by autoradiography.


Figure 4. Elongation rates of the unmodified and the Cys883 mutant T7 RNA polymerase. (A) Elongation rate assay at 5 mM Mg2+ with unmodified polymerase (T7 RNAP) and the Cys883 mutant. The stalled polymerase samples were elongated and stopped every second from 6 (or 8 for the mutant) to 17 s as indicated by numbers or dots and transcripts were separated in a 5% polyacrylamide-7 M urea gel and subjected to autoradiography. A denatured 5[prime]-32P-labelled 100 bp DNA ladder was co-electrophoresed with the transcripts (M) and sizes in nucleotides are given to the left (the denatured DNA fragments resulted in double bands). (B) Elongation rate assay as in (A), but at 1 mM Mg2+ and with time points every 10 s from 30 (or 40 for the mutant) to 120 s as indicated by numbers and dots. (C) Transcript length plotted against duration of transcription. Transcript lengths, determined from the gels shown in (A) and (B), are indicated as squares for the unmodified polymerase (wt) and circles for the Cys883 mutant at 5 (open symbols) and 1 mM Mg2+ (filled symbols). Notice that the transcripts extrapolate to ~25 nt (the size of the stalled transcript) at the 0 time point.

T7 RNA polymerase modified at the C-terminal carboxy group exhibits a magnesium ion-dependent decrease in the elongation rate

We then compared the elongation rate of the Cys883 mutant with those of two C-terminal carboxy group-modified polymerases, i.e. the Cys-OMe883 and decarboxy-Cys883 derivatives (Fig. 2B). Since T7 RNA polymerase is a processive polymerase it is possible, using the elongation rate assay described above, to compare directly the elongation rates in mixing experiments where the Cys883 polymerase is mixed with one of the C-terminal carboxy group-modified polymerases. This approach ensures that the polymerases are subjected to exactly the same conditions. The results of the elongation rate assays at 5 and 1 mM Mg2+ are depicted in Figure 5 and the calculated elongation rates from three independent experiments are listed in Table 1. At 5 mM Mg2+, one band is observed at each time point in Figure 5A and B (left), so no significant difference between the elongation rates of the three polymerase derivatives exists. In contrast, at 1 mM Mg2+ two separate bands at each time point in Figure 5A and B (right) occur, implying that both the Cys-OMe883 and the decarboxy-Cys883 derivatives exhibit an impaired elongation rate when compared with the Cys883 polymerase. These results were corroborated in at least three independent mixing experiments and in reactions with separate polymerase derivatives. Moreover, the effects were not ionic strength-dependent, since KCl could be added to 150 mM without affecting the relative elongation rates and the polymerase derivatives produced a similar amount of full-length transcript (data not shown). Elongation rate experiments were also performed at 2 and 0.5 mM Mg2+ and they confirmed that the elongation rate of the C-terminal modified polymerases in relation to that exhibited by the Cys883 polymerase decreased with decreasing magnesium ion concentration (Table 1).

DISCUSSION

Divalent metal ions are crucial in biological phosphoryl transfer reactions, both as activators of nucleophiles and as stabilizers of pentacovalent phosphate transition states and anionic leaving groups. In the case of polymerases, a large body of evidence supporting a two metal ion catalytic mechanism has accumulated in recent years. This mechanism, which was originally proposed for DNA polymerase I from E.coli, involves two magnesium ions at the catalytic centre, one of which might activate the attacking 3[prime]-OH of the primer and the other stabilize the pentacovalent phosphate transition state of the reaction (16,32). The catalytic metal ions are coordinated by substrate phosphates and acidic side chains in the polymerases. In T7 RNA polymerase, Asp537 and Asp812 are positioned at the active site (13) and these two acidic residues are invariant in known polymerases. In contrast, a third acidic side chain positioned near the catalytic centre in polymerase structures is not found in T7 RNA polymerase. However, examination of the T7 RNA polymerase structure reveals that a third carboxy group is positioned near the active site, namely the C-terminal carboxylate (Fig. 1). This carboxylate is not superimposable on the `third' acidic residue in the other polymerases, but previous work has shown that the integrity of the hydrophobic C-terminal -Phe-Ala-Phe-Ala-OH883 `foot' is important for activity (27,28). Moreover, crystallographic data indicate that the `foot' is poorly ordered. Therefore, we examined whether the flexible C-terminal carboxy group is important for metal ion-dependent catalysis by T7 RNA polymerase.


Figure 5. Elongation rate mixing experiments with C-terminal-modified polymerases. (A) The Cys883 polymerase was mixed with the methyl ester derivative (Cys-OMe883) at a 1:3 ratio at 5 (left) or 1 mM Mg2+ (right) and elongation assays were performed as shown in Figures 3 and 4. Elongations were stopped after the indicated time points. At 1 mM Mg2+, the Cys-OMe883 derivative is trailing, which results in two bands at each time point, indicated for the 240 s lane to the right. (B) The same experiment as in (A) but the Cys883 polymerase was mixed with the decarboxy-Cys883 derivative at a 2:1 ratio. At 1 mM Mg2+, the decarboxy-Cys883 derivative is trailing, which is indicated for the 120 s time point to the right.

If the C-terminal carboxy group is important for catalytic metal ion binding, it is expected that a carboxylate-modified enzyme would exhibit a lower affinity for magnesium ions. This will result in a magnesium ion-dependent decrease in the elongation rate relative to that of the unmodified enzyme. In order to investigate the importance of the C-terminal carboxy group, we used a recently developed intein-based protein purification system to introduce a methyl ester and a decarboxylated C-terminus of T7 RNA polymerase (Fig. 2). The intein-based approach required an Ala883->Cys883 mutation, which resulted in a polymerase mutant with an elongation rate of 88-90% of the unmodified enzyme at both high (5 mM) and low (1 mM) magnesium ion concentrations. When modifications of the C-terminal carboxylate of the Cys883 mutant were introduced, a further decrease in the elongation rate was observed at low Mg2+ concentrations, whereas the modified enzymes were unaffected at 5 mM Mg2+. Therefore, the modified enzymes exhibit a lower affinity for one or both of the catalytic metal ions. Since the elongation rate assays of the modified enzymes did not reveal an increase in premature termination, the slower rates reflect a decreased catalytic efficiency of phosphoester bond formation rather than a decreased degree of processivity.

Although the C-terminal carboxylate is important for catalysis by T7 RNA polymerase, it is not as critical as Asp537 and Asp812, since mutations at the latter positions lead to enzymes with a 25-50 000-fold reduction in catalytic activity (21). However, it is striking that the C-terminal sequence is highly conserved among phage RNA polymerases and that the position of the C-terminus is absolutely conserved (27). In T7 RNA polymerase, the distance between the C[alpha] atoms of Asp537 and the C-terminal Ala883 is ~6.3 Å (13) and superimposing T7 RNA polymerase on the Mg2+-containing T7 DNA polymerase structure suggests that the C-terminal C[alpha] atom is ~10 Å from the metal ions (data not shown). Since this places the C-terminal carboxylate oxygens at least 7.5 Å from the nearest magnesium ion, the most obvious explanation is that the C-terminal carboxy group is important for the local structure of the active site. This could be achieved by interacting with the Asp537-containing [beta]-sheet, which is very close to the C-terminus (Fig. 1). Although this seems the most likely explanation, T7 RNA polymerase has been crystallized in the absence of substrates, so a minor structural rearrangement could bring the flexible C-terminus of an elongating polymerase closer to the metal ions. Therefore, it is also possible that the C-terminal carboxylate is directly involved in coordinating one of the catalytic metal ions.

ACKNOWLEDGEMENTS

Bo Porse is thanked for supplying the pUT7-L11-RNA1 plasmid and critically reading the manuscript. This study was supported by the Danish Research Council Biotek II Programme.

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*To whom correspondence should be addressed at: Department of Biological Chemistry, University of Copenhagen, Sølvgade 83 H, DK-1307 Copenhagen K, Denmark. Tel: +45 353 22008; Fax: +45 353 22040; Email: janchr@mermaid.molbio.ku.dk

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G. M. Cheetham and a. T. A. Steitz
Structure of a Transcribing T7 RNA Polymerase Initiation Complex
Science, December 17, 1999; 286(5448): 2305 - 2309.
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