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Short Communication |
6 RNA-dependent RNA polymerase
1 Institute of Biotechnology and Department of Biological and Environmental Sciences, Viikki Biocenter, PO Box 56 (Viikinkaari 5), 00014 University of Helsinki, Finland
2 Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN, UK
Correspondence
Dennis H. Bamford
dennis.bamford{at}helsinki.fi
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ABSTRACT |
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6 is capable of primer-independent initiation, as are many RNA polymerases. The structure of this polymerase revealed an initiation platform, composed of a loop in the C-terminal domain (QYKW, aa 629–632), that was essential for de novo initiation. A similar element has been identified in hepatitis C virus RNA-dependent RNA polymerase. Biochemical studies have addressed the role of this platform, revealing that a mutant version can utilize a back-priming initiation mechanism, where the 3' terminus of the template adopts a hairpin-like conformation. Here, the mechanism of back-primed initiation is studied further by biochemical and structural methods.
These authors contributed equally to the work. 
The coordinates and structure factors of the SG mutant model have been deposited in the Protein Database with accession codes 1WAC and R1WAC, respectively.
Information on data collection and refinement statistics is available as supplementary material in JGV Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Kao et al., 2001; van Dijk et al., 2004). A previously characterized 6 mutant polymerase was found to be prone to back-primed initiation, where the 3' end of the template loops back to form a hairpin-like structure that is subsequently extended by the polymerase (Laurila et al., 2002). Hepatitis C virus (HCV) polymerase and the related bovine viral diarrhea virus (BVDV) polymerase are capable of de novo initiation of RNA synthesis (Ranjith-Kumar et al., 2002a, b; Sun et al., 2000; Zhong et al., 2000b). However, in vitro, these enzymes preferentially utilize a back-priming initiation mode (Behrens et al., 1996; Luo et al., 2000; Zhong et al., 1998, 2000a). This type of initiation is deleterious in vivo, as the newly produced daughter strand remains bound covalently to the template strand (Kao et al., 2001). Several factors have been proposed to prevent back-primed initiation in 6 polymerase: stabilization of the initiation complex by a primer-mimicking loop (Butcher et al., 2001) and a high concentration of initiatory nucleotides (Laurila et al., 2002). A recent study (Ranjith-Kumar et al., 2003) found that similar factors are important for HCV polymerase to initiate in a primer-independent mode in vitro.
The RdRP structures solved to date show the canonical right hand-like structure with finger, palm and thumb subdomains, similar to the DNA-dependent RNA polymerases, reverse transcriptases and DNA-dependent DNA polymerases (Cheetham & Steitz, 2000; Doublie et al., 1999; Ollis et al., 1985). High-resolution structures of HCV, 6 and BVDV RdRPs (Ago et al., 1999; Bressanelli et al., 1999, 2002; Butcher et al., 2001; Choi et al., 2004; Lesburg et al., 1999; Salgado et al., 2004) revealed strong similarities (but little sequence identity), suggesting an evolutionary link (Butcher et al., 2001; Koonin, 1991). For these enzymes, a structural element positions the 3' end of the template RNA for proper primer-independent initiation. The C-terminal domain of 6 polymerase contains a stabilizing loop – QYKW (residues 629–632) – with Tyr630 establishing base-stacking interactions with the template and incoming GTPs (Butcher et al., 2001). In HCV polymerase, a 
-hairpin protruding from the thumb domain and a C-terminal hydrophobic pocket form the proposed stabilizing platform (Hong et al., 2001; Ranjith-Kumar et al., 2003).
Previous biochemical studies (Laurila et al., 2002) addressed an initiation-platform mutant; however, low yields of purified protein precluded structural studies. To obtain a functionally similar mutant for crystallization trials, the four bulky amino acids QYKW (residues 629–632) were changed to small residues, SG, by site-directed mutagenesis. For expression of the wild-type 6 polymerase, plasmid pEMG2 was used (Makeyev, 2001); this was then used to construct the SG mutant plasmid. Firstly, a short fragment of the 6 polymerase gene was PCR-amplified by using oligonucleotides 5'-CCAGTTCAGCCCTGAGTACGGTGT-3' and 5'-GCCATGCATCAGTACCTCGTGGATATTCGCCGAGACATCGGCCTCGGTACCGGAGAGTTTGTT-3' as upstream and downstream primers, respectively. The PCR product was digested with NruI–NsiI and ligated with similarly cut pEMG2. The insert in the resultant plasmid pRT2 [QYKW(629–632)SG mutant] was verified by sequencing. The mutant protein (denoted SG) migrated similarly to the wild-type (WT) in SDS-PAGE (Fig. 1a). Expression and purification were done as described previously (Makeyev & Bamford, 2000a), with gel filtration (Superdex 75 16/60) as a final purification step for the mutant.
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; Yang et al., 2001) and analysed by gel electrophoresis (Makeyev & Bamford, 2000a; Pagratis & Revel, 1990) in 1·2 % agarose gels, followed by autoradiography. ssRNA templates (Fig. 1c
) were produced in vitro by run-off transcription with T7 RNA polymerase (Makeyev & Bamford, 2000a, 2001). The first (s
+ ssRNA) template resembles 6 small genomic segment (s+), but contains an extensive internal deletion (Makeyev & Bamford, 2000b) (Fig. 1c). Its 5 nt single-stranded 3' tail apparently does not form any tertiary structure (Mindich et al., 1994) and is long enough to be delivered to the active site of the enzyme (Butcher et al., 2001). The SG mutant could not utilize this substrate efficiently, whereas the WT control reaction produced a readily detectable amount of full-length double-stranded RNA (dsRNA) product (Fig. 1b, lanes 1 and 2). The second template, s+13, is a derivative of s+ RNA with a 13 nt extension, CUAGAGGAUCCCC-3', that can form a transient hairpin structure to prime RNA synthesis (Fig. 1c) (Laurila et al., 2002). Both the WT and SG polymerases readily accepted this template, producing full-length dsRNA products equally well (Fig. 1b, lanes 3 and 4). The reduction in the WT reaction products using the s+ template is due to the 3'-terminal U, which is less favoured than the 3'-terminal C in s+13 (Makeyev & Bamford, 2000b; Yang et al., 2001). The third ssRNA substrate, s+HP, with a preformed hairpin loop (CUAGGGGUUCGCCCC-3') at the 3' terminus (Fig. 1c) (Laurila et al., 2002), was replicated approximately fourfold more efficiently by the SG mutant than by the WT (as quantified from Fig. 1b, lanes 3 and 4). Thus, the SG mutant has a preference for templates with a 3' hairpin loop, whereas the WT prefers non-looped ssRNA 3' templates. It is also noted that, for SG, the tetralooped template s+HP is somewhat less favoured when compared to s+13 (Fig. 1b, lanes 4 and 6). We interpret this as being due to more restricted entrance of the bulkier 3' terminus of s+HP to the active site.
The initiation mode of the SG polymerase was studied further by using denaturing gel electrophoresis to analyse the polymerase reaction products (Fig. 2a). The complementary strands of de novo-produced duplex RNA migrate as single strands under denaturing conditions, whereas hairpin-like dsRNAs are not separated, migrating as dsRNA molecules (Fig. 2a). Polymerase reactions were stopped by adding 40 mM EDTA, gel-filtrated (G-50; Amersham Biosciences), vacuum-dried and dissolved in H2O. Denaturing agarose gel electrophoresis was executed as described by Sambrook et al. (1989), followed by autoradiography. As expected, in the presence of 2 mM MnCl2 and 5 mM MgCl2 (optimal divalent cation conditions), the WT-produced daughter strands were almost completely separable from the s+13 template upon denaturation (Fig. 2b, lane 1). In contrast, over half of the replication products of the SG mutant could not be converted into the single-stranded form (Fig. 2b, lane 4), indicating that this platform mutant is prone to back-primed initiation. The nature of the hairpin-like product (Fig. 2b) was confirmed by comparing it to a corresponding product of the previous, equivalent initiation-platform mutant YKW(630–632)GSG (Laurila et al., 2002).
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, lanes 2 and 5), but did not affect the mode of initiation. In the absence of manganese (but with an identical total divalent cation concentration), SG lost practically all its residual capacity to initiate de novo (Fig. 2b, compare lanes 4 and 6), with 
40 % reduction in overall activity. Under these conditions, the amount of dsRNA products synthesized by the WT decreased by 75 %, with no change in the initiation mode. The stimulatory effect of manganese has been observed previously (Makeyev & Bamford, 2000a) and its effect on initiation was studied by using [
-32P]GTP. This nucleotide is only incorporated into de novo-initiated RNA chains. As expected, the WT is relatively inefficient without Mn2+, but the efficiency of RNA synthesis increased considerably with increasing Mn2+ concentration (Fig. 2c), whilst for the SG mutant, de novo-initiated synthesis did not respond to Mn2+ stimulus until the concentration reached 0·5 mM.
For structural studies, the protein was buffered by using 10 mM Tris/HCl (pH 8·0) with 100 mM NaCl, concentrated to 4 mg ml–1 and crystallized by sitting-drop vapour diffusion in 0·1 M sodium citrate (pH 5·6), 19 % 2-propanol, 19 % polyethylene glycol 4000, 5 % glycerol. We were unable to obtain co-crystals with RNA or nucleotides. X-ray diffraction data were collected at ID29 at the European Synchrotron Radiation Facility, Grenoble, France, by using an ADSC Q210 detector, 1° oscillation images, at 100 K [with 25 % (v/v) glycerol as cryoprotectant] and processed with HKL-2000 (Otwinowski & Minor, 1997). The SG mutant crystals are not isomorphous with WT crystals (Butcher et al., 2001; Salgado et al., 2004), belonging to space group P21 with unit-cell dimensions of a=76·6 Å, b=105·9 Å, c=157·7 Å, =98·8°. Molecular replacement with CNS (Brunger et al., 1998) used the WT protein as a search model. Absence of electron density in the 2|Fo|-|Fc| map in the mutated loop region and strong negative electron-density features (–10
) in the averaged-difference Fourier map (Fig. 3b) after rigid-body refinement (Rfactor=34·4 %, Rfree=34·3 %) of the three molecules in the asymmetric unit show that the structure of the initiation loop has been disrupted. Mutated residues were modelled by using CALPHA (Esnouf, 1997) and the monomer was refined with CNS, imposing threefold non-crystallographic constraints (Rfactor=24·1 %, Rfree=28·0 %). Statistics for the model and refinement are given as supplementary material in JGV Online.
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; Salgado et al., 2004). Furthermore, the catalytic site defined by the aspartic residues 324, 453 and 454 does not appear to be perturbed, in accord with the biochemical data (Fig. 2b), showing that the mode of initiation, but not catalysis, is affected by the mutation (Fig. 1b).
The electron density for the residues upstream and downstream of the mutated loop is poorly defined. This is associated with an expansion in the active-site cavity in the mutant enzyme, due to a loss of stabilizing interactions. In the WT, residues 616–619 of the C-terminal domain pack against residues 305–308 of the palm domain, whereas, in the mutant, these regions shift apart by 1·4 Å, creating a slight gap and destabilizing the whole C-terminal domain (the ratio of mean crystallographic B-factor, a measure of disorder, between the C-terminal domain and the rest of the protein is 2·3 for SG, compared to 1·5 for WT). The effect extends to side chains of contact residues, which are rearranged and generally less well-defined in the SG mutant (Fig. 3d). It is likely that a major reason for the destabilization is the loss of the bulky side chain of W632, which, in the WT, makes bridging contacts with the 305–308 loop region of the palm domain (Fig. 3d). We suggest that this overall destabilization is significant and that the initiation platform plays important roles both in stabilizing the template and in modulating the switch from initiation to elongation via its interactions with the surrounding protein residues.
Finally, in contrast to the WT enzyme, where manganese ions are bound even in the absence of Mn2+ in the crystallization conditions (P. S. Salgado, D. I. Stuart & J. M. Grimes, unpublished data), the electron-density map for the SG mutant shows no evidence of bound manganese, suggesting that the stimulatory effect of manganese (Fig. 2b and c) on de novo initiation in the SG mutant might arise from induced ordering of the C-terminal domain.
It is possible that truncation of the C terminus of HCV polymerase could produce the same disruption of the initiation platform as is seen for the SG mutation, explaining its preference for back-primed initiation in vitro (Behrens et al., 1996; Luo et al., 2000; Zhong et al., 1998, 2000a). These results strengthen our view of the polymerase structure as switchable, with a discrete set of contacts stabilizing the initiation-competent form of the enzyme so that relatively modest changes can have long-range effects, controlling the switch from the initiation to elongation phase, with premature conformational switching producing a structure that preferentially initiates by back-priming.
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ACKNOWLEDGEMENTS |
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Received 31 July 2004;
accepted 21 October 2004.
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