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Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8501, Japan
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Abstract |
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Introduction |
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A prominent case of elongation control involves three transcription elongation factors, namely DRB sensitivity-inducing factor (DSIF), negative elongation factor (NELF) and positive elongation factor (P-TEF) b. DSIF and NELF together bind to Pol II and repress transcription elongation in a promoter-proximal region (Wada et al. 1998a; Yamaguchi et al. 1999a). The kinase P-TEFb then reverses the repression by phosphorylating the C-terminal domain (CTD) of the largest subunit of Pol II and by facilitating the dissociation of NELF (Wada et al. 1998b; Yamaguchi et al. 1999a), after which DSIF is, instead, able to activate transcription elongation (Bourgeois et al. 2002; Yamada et al. 2006).
Hepatitis delta virus (HDV) is a defective virus that requires the helper function of hepatitis B virus (HBV) to proliferate and also potentiates its pathogenic effects (reviewed in Lai 2005; Taylor 2006). HDV has the ~1.7 kb single-stranded RNA genome that forms a partially double-stranded "rod-like" structure, and unlike many other RNA viruses that replicate using their own RNA polymerases, replication and transcription of HDV are considered to proceed via RNA-dependent RNA synthesis by Pol II (Filipovska & Konarska 2000; Moraleda & Taylor 2001; Chang et al. 2006; Greco-Stewart et al. 2007). Hepatitis delta antigen (HDAg), the only protein encoded by HDV, is critical for these processes. Two isoforms of HDAg with different C-termini, HDAg-S (195 amino acid) and HDAg-L (214 amino acid), are generated by RNA editing, and the shorter isoform is considered to be mainly involved in HDV replication and transcription (Chao et al. 1990). HDAg is an RNA-binding protein rich in basic amino acid residues and forms viral ribonucleoprotein complexes by binding to partially double-stranded HDV RNA.
The surprising fact is that the cellular DNA-dependent RNA polymerase carries out the RNA-dependent RNA synthesis. Of possible relevance to it, we have shown that HDAg-S (hereafter simply called HDAg) directly binds to Pol II and stimulates both DNA- and RNA-templated transcription by enhancing the elongation step in vitro (Yamaguchi et al. 2001, 2002). This study was initially prompted by the finding that HDAg has a weak sequence similarity to an N-terminal segment of NELF-A, the largest subunit of NELF. Since the same region of NELF is also implicated in Pol II binding (Narita et al. 2003), it is very likely that the regions of similarity form a conserved structure that recognizes a common surface on Pol II. Biochemical studies suggested that HDAg stimulates transcription elongation by two different mechanisms: (i) HDAg reverses the negative effect of DSIF and NELF by displacing NELF from Pol II, and (ii) the HDAg-Pol II interaction itself further enhances transcription elongation (Yamaguchi et al. 2001). More recently, high-resolution kinetic analysis suggested that HDAg accelerates forward translocation of Pol II (Nedialkov et al. 2003). Identification of HDAg as a "viral" transcription elongation factor immediately led us to speculate that HDAg may contribute to the unusual RNA-dependent RNA synthesis by Pol II.
Hence, more detailed studies on HDAg will provide useful insights not only into the viral life cycle but also into the basic mechanism of Pol II transcription. In addition, HDAg will serve as a model of cellular transcription elongation factors, especially of NELF, as HDAg is small in size and easy to manipulate, compared to the four-subunit NELF complex. Here, using site-specific photocrosslinking and transcription with reconstituted elongation complexes, we present evidence that HDAg interacts with the clamp of Pol II and affects transcriptional fidelity. Together with our previous findings, we propose a model in which HDAg interacts with and loosens the clamp, and thereby accelerates forward translocation of Pol II at the cost of fidelity.
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Results |
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HDAg is a 195-amino acid protein with a modular structure (see Fig. 2A). A coiled-coil sequence, which is responsible for oligomerization (Zuccola et al. 1998), resides within the N-terminal one-third of the protein. HDAg also has a nuclear localization signal (NLS) and two stretches of arginine-rich motifs (ARM) that are involved in RNA binding. Previously, the C-terminal 66 amino acid segment of HDAg was shown to be sufficient for its binding to human Pol II (Yamaguchi et al. 2001). We initiated this study by characterizing the Pol II-HDAg interaction in more detail. In the first set of experiments, an oligomerization-deficient mutant of HDAg (
N88) fused to glutathione S-transferase (GST) and Flag- and His-tagged human Pol II (FH-Pol II) were individually prepared, combined and subjected to glutathione Sepharose affinity chromatography. As shown in Fig. 1A, a fraction of FH-Pol II co-precipitated with GST-HDAgN88. This interaction is likely to be direct, as both Pol II and HDAg used were affinity-purified under stringent conditions. This assumption was further validated by carrying out GST pull-down assays in the presence of RNase A and DNase I, which had no appreciable effect on the amount of co-precipitated Pol II (data not shown). From these results, it can be concluded that HDAg interacts with Pol II directly and efficiently under these conditions.
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Amino acid sequences of the C-terminal region of HDAg, deduced from ~200 clinical isolates of HDV, are compared in Fig. 2A. To determine HDAg's amino acid residues that are important for its interaction with Pol II, we mutated evolutionarily conserved or non-conserved amino acids in the HDAg C-terminal region to Ala or Cys, respectively. GST pull-down assays demonstrated that HDAg's conserved amino acid residues such as Asp-187, Ile-188 and Phe-194 are critical for the interaction, while its non-conserved amino acid residues such as Ser-170, Ala-180 and Thr-182 are not (Fig. 2B). Besides, addition of an extra Cys residue to the extreme C-terminus of HDAg (196C) did not affect the interaction substantially (Fig. 2B). The finding that the amino acid residues critical for the HDAg-Pol II interaction are conserved among the clinical isolates of HDV supports the idea that this interaction is important to the viral life cycle.
Both Rpb1 and Rpb2 are in close proximity to the C-terminal region of HDAg
To map a Pol II region involved in HDAg binding, we used N-((2-pyridyldithio)ethyl)-4-azidosalicylamide (AET), a heterobifunctional cross-linker containing a photoactivatable azido group and a thiol-reactive pyridyldithio group that are separated by a 15 Å linker arm (Chen et al. 1994; Niu et al. 1996; Chen & Hahn 2003, 2004). Since authentic HDAg has no Cys, the cross-linker can be introduced into HDAg at a desired position by substituting or adding Cys. Thus, radioiodinated AET was first attached to His-tagged, full-length HDAg through a Cys residue appended to the C-terminus (196C), and each modified protein was incubated with a crude Pol II fraction, after which the cross-linker was activated by a brief exposure to UV light. The samples were then treated with 2-mercaptoethanol (2ME), which cleaves disulfide bonds to allow the transfer of the radiolabeled cross-linker to photocrosslinked proteins, and Pol II was immunoprecipitated and analyzed by SDS-PAGE. As a result, HDAg 196-AET was photocrosslinked to Rpb1 and Rpb2, but not to the other small subunits of Pol II (Fig. 3A). These photocrosslinks seem to be specific, as they were abolished by the Pol II-binding defective I188A mutation (Fig. 3A).
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N20, which lacked part of the N-terminal oligomerization domain. N20 was indeed monomeric, judging from gel filtration and glutaraldehyde cross-linking studies (data not shown). Of the four Cys derivatives that we examined, N20 carrying the cross-linker at the extreme C-terminus was photocrosslinked to both Rpb1 and Rpb2 (Fig. 3B), which is consistent with the previous finding and supports the stochastic model of photocrosslinking. Interestingly, N20 carrying the cross-linker at different positions gave different results. The cross-linker placed at position 182 was transferred to only Rpb1, whereas the cross-linker placed at position 170 or 180 was not transferred to any of the Pol II subunits appreciably (Fig. 3B), demonstrating that the photocrosslinking reactions are highly position-specific. These results lead us to conclude that both Rpb1 and Rpb2 are in close proximity to the C-terminal region of HDAg within the functional HDAg-Pol II complex. HDAg interacts with the Pol II clamp
To determine the sites on Rpb1 and Rpb2 to which photocrosslinking occurred, we performed proteolytic mapping with hydroxylamine, which cleaves between Asp and Gly residues. After photocrosslinking with HDAgN20 182-AET or 196-AET, radiolabeled Rpb1 and Rpb2 were resolved by SDS-PAGE, recovered from gel slices, digested with hydroxylamine, and subjected to second SDS-PAGE (Fig. 4). Silver staining of the materials revealed complex patterns of protein ladders due to incomplete digestion with hydroxylamine. Whichever HDAg derivative was used, hydroxylamine digestion of Rpb1 resulted in a strongly radiolabeled band of 35 kDa and some weakly radiolabeled bands, sizes of which were consistent with the assumption that these bands contained the N-terminal 296 amino acid segment (positions 1–296) of Rpb1 (Fig. 4A). This assumption was confirmed by immunoblotting of the hydroxylamine-treated materials with an antibody against the extreme N-terminal peptide of Rpb1 (Fig. 4A). As for Rpb2, its hydroxylamine digestion resulted in a strongly radiolabeled band of 16 kDa and some weakly radiolabeled bands, sizes of which were consistent with the assumption that these bands contained the C-terminal 144 amino acid segment (positions 1031–1174) of Rpb2 (Fig. 4B). This assumption was confirmed by immunoblotting with an antibody against the extreme C-terminal peptide of Rpb2 (Fig. 4B).
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HDAg affects recognition of incoming nucleotides by Pol II
Since the clamp module is located near the catalytic center and is known to hold template DNA and nascent RNA in place, it is tempting to speculate that the clamp binding has direct implications in the role of HDAg. With the aim to understand the relevance of the clamp binding, we examined the possibility that HDAg may have an impact on transcriptional fidelity. For this purpose, we reconstituted elongation complexes with purified Pol II and synthetic oligonucleotides (Fig. 5A) by a recently developed procedure (Komissarova et al. 2003). Before carrying out this study, we confirmed using a conventional dC-tailed template that FH-Pol II used in this study (Fig. 1A) is functionally active and is subject to regulation by HDAg (Fig. 5B).
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Next, we aimed to determine whether HDAg has an impact on transcriptional fidelity, specifically on the recognition of incoming nucleotides by Pol II. Since transcription based on canonical base pairing proceeds with high fidelity, nucleotide mis-incorporation is difficult to quantify under normal circumstances. We thus carried out transcription with a DNA template containing 8-oxoguanine (8-oxoG) in the template strand as a convenient assay to evaluate transcriptional fidelity. 8-oxoG is a non-canonical base normally generated by oxidative damage of guanine residues in the genome, and we reported previously that Pol II incorporates two different nucleotides, CTP and ATP, opposite 8-oxoG with comparable efficiency (Kuraoka et al. 2003). Consistent with our previous finding (Kuraoka et al. 2003), a strong pause was observed at the 8-oxoG site, and only a small fraction of the elongation complexes reached the 3' end of the template even at high concentrations of NTPs (Fig. 5C). HDAg had only a modest effect on the production of the full-length transcript (Fig. 5C). To figure out which nucleotide was incorporated opposite 8-oxoG under these conditions, we amplified the transcripts by RT-PCR, cloned the PCR products, and determined the sequences of a number of independent clones. As shown in Fig. 5D, ATP and CTP, but not GTP or UTP, were incorporated opposite 8-oxoG at various ratios. In the absence of HDAg, ATP was preferentially incorporated over CTP, and the ATP : CTP ratio was similar at low and high concentrations of NTPs. Strikingly, HDAg increased incorporation of CTP such that the ATP : CTP ratio was reversed. When Mn2+, which is considered to reduce transcriptional fidelity (Huang et al. 1997), was used instead of Mg2+ as a divalent cation during RNA synthesis, incorporation of CTP was again dominant over incorporation of ATP. In contrast, TFIIS, which is considered to enhance transcriptional fidelity (Jeon & Agarwal 1996; Thomas et al. 1998), increased the incorporation of ATP. Thus, HDAg clearly has an impact on which nucleotide is incorporated under these conditions. Given the assumption that 8-oxoG:A and 8-oxoG:C are recognized as match and mismatch base pairs, respectively, by Pol II, it follows that HDAg reduces transcriptional fidelity.
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Discussion |
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HDAg has a limited sequence similarity to the N-terminal ~200 amino acid region of NELF-A, the largest subunit of NELF (Yamaguchi et al. 2001). Since the same region of NELF-A is also implicated in Pol II binding (Narita et al. 2003), it is very likely that the regions of similarity serve as structurally similar Pol II-binding domains. Hence, the present findings imply that NELF also interacts with the Pol II clamp and thereby inhibits, rather than stimulates, transcription elongation. It would then be interesting to know why HDAg and NELF exert opposite functions through similar Pol II-binding domains.
Among a number of Pol II-binding proteins, yeast TFIIB, TFIIF and TFIIS have been characterized for their association with yeast Pol II at molecular resolution. The N-terminal domain of TFIIB interacts with the dock domain and active site of Pol II near the RNA exit channel, whereas the location of the CTD of TFIIB remains elusive because of inconsistency between biochemical and X-ray structural data (Chen & Hahn 2003, 2004; Bushnell et al. 2004). As for TFIIF, the Tfg1 subunit interacts with the Rpb4 and Rpb7 subunits and the clamp, whereas the Tfg2 subunit interacts with an extended region along the active site cleft of Pol II (Chung et al. 2003). As for TFIIS, its domain II interacts with the jaw domain of Pol II, whereas the interdomain linker and the domain III extend into the funnel and pore near the active site of Pol II (Kettenberger et al. 2003, 2004). From these structural data, it can be inferred that HDAg, NELF and TFIIF may interact with overlapping regions of Pol II. This assumption is consistent with the observation that the negative effect of NELF on transcription elongation is reversed by increasing concentrations of TFIIF (Renner et al. 2001).
Effect of HDAg on transcriptional fidelity
Transcriptional fidelity is normally high, and nucleotide mis-incorporations opposite canonical bases are very rare events that are difficult to quantify. In an elegant study with bacterial RNA polymerase, elongation complexes stalled at a discrete position were incubated with a complementary or non-complementary NTP, and from the kinetics of one nucleotide addition, mis-incorporation frequency was calculated to be 3.8 x 10–8 to 5.9 x 10–6 (Foster et al. 2001). Although we carried out similar experiments with human Pol II, we could not detect mis-incorporation but instead observed incorporation of correct bases, probably due to impurities in commercial preparations of NTPs (data not shown). We thus changed our strategy and used DNA template containing 8-oxoG, a non-canonical base that is normally generated by oxidative damage of guanine residues in the genome. 8-oxoG is highly mutagenic because DNA polymerases incorporate not only dCTP but also dATP opposite 8-oxoG (Shibutani et al. 1991). Similarly, during transcription, Pol II incorporates CTP and ATP opposite 8-oxoG with comparable efficiency, leading to "transcriptional mutagenesis" (Kuraoka et al. 2003). Therefore, the use of 8-oxoG-containing template provides a convenient way to study factors that influence the recognition of incoming nucleotides.
An important question relevant to this study is incorporation of which nucleotide should be considered "mis-incorporation." Structural studies of bacterial DNA Pol I showed that 8-oxoG in the anti conformation pairs with cytosine and is recognized as a mismatch, whereas 8-oxoG in the syn conformation pairs with adenine and mimics a canonical base pair (Hsu et al. 2004). To examine whether Pol II, like bacterial DNA Pol I, discriminates between 8-oxoG:C and 8-oxoG:A base pairs, we constructed and analyzed in silico the structures of yeast Pol II elongation complexes bearing 8-oxoG at position i + 1 in the template strand. Molecular modeling suggested that 8-oxoG in the syn conformation is energetically more favorable than that in the anti conformation within the Pol II active center (Fig. 6B; see Experimental procedures). When adopting the anti conformation, 8-oxoG appears to induce template distortion in order to avoid a potential steric clash between the C8 oxygen of 8-oxoG and adjacent sugar and phosphate moieties of DNA. This distortion may cause the pair of 8-oxoG(anti) and cytosine to be recognized as a mismatch by Pol II. This idea is also supported by the observation (Fig. 5D) that formation of the 8-oxoG:C base pair was increased or decreased by Mn2+ and TFIIS, respectively, which are known to affect transcriptional fidelity in opposite directions (Huang et al. 1997; Thomas et al. 1998). Thus, given the assumption that 8-oxoG:C is recognized as a mismatch, it then follows that HDAg reduces transcriptional fidelity.
Potential physiological role of HDAg binding to the Pol II clamp
In summary, we have shown evidence that HDAg interacts with the Pol II clamp and affects transcriptional fidelity in vitro. These functions of HDAg may directly contribute to the life cycle of HDV in vivo. We offer the following perspective that is consistent with this view. HDV has a single-stranded circular ~1.7 kb RNA genome with an unbranched rod-like structure (reviewed in Taylor 2006). Unlike many other RNA viruses, HDV does not encode its own RNA polymerase, but instead its genome replication and transcription are considered to proceed via RNA-dependent RNA synthesis by Pol II (Filipovska & Konarska 2000; Moraleda & Taylor 2001; Chang et al. 2006; Greco-Stewart et al. 2007). It is then tempting to speculate that through binding to the clamp, HDAg may not only affect discrimination of incoming nucleotides but also affect template recognition by Pol II and thereby facilitate the unusual RNA-dependent RNA synthesis (Fig. 6A).
This model is based on many assumptions. For example, it has not been examined whether HDAg has any effect on template recognition by Pol II. This issue may be studied by preparing elongation complexes undergoing RNA-templated transcription from purified Pol II and RNA oligonucleotides. In addition, although HDAg accelerates Pol II transcription in part through competition with the NELF in vitro, it has not been determined whether this holds true in vivo during HDV proliferation. HDAg is the only HDV protein that is considered to play various roles during the viral life cycle, and this multifunctionality makes the analysis of HDAg complicated. Perhaps, however, with the Pol II binding-deficient point mutants of HDAg identified in this study, we may be able to study the relevance of Pol II binding by HDAg. Other unanswered questions include how Pol II is recruited to the HDV genome, how Pol II initiates RNA synthesis, and what the transcription complexes involved are composed of. Thus, many issues need to be addressed to validate our model and to precisely understand the mechanism of RNA-dependent RNA synthesis of HDV, which will be the subject of future study.
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Experimental procedures |
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Point mutations were introduced by PCR using appropriate mutagenic primers. For S170C, the 170th codon was changed from AGC to TGC. For A180C, the 180th codon was changed from GCT to TGT. For T182C, the 182nd codon was changed from ACC to TGC. For D187A, the 187th codon was changed from GAC to GCC. For I188A, the 188th codon was changed from ATA to GCA. For F194A, the 194th codon was changed from TTC to GCC. For 196C, the termination codon was replaced by the sequence TGTTGA. For expression of GST fusion proteins, PCR products were cloned into BamHI and NotI sites of pGEX-6P-1 (GE Healthcare), and all the insert sequences were verified. For expression of His-tagged proteins, the inserts were subcloned into NdeI and BamHI sites of pET-14b (Novagen). All the HDAg proteins were expressed in Epicurian Coli BL21-CodonPlus (DE3)-RIL cells (Stratagene). For photocrosslinking study, His-HDAg proteins were first purified from bacterial lysates on a phosphocellulose column as described (Sheu & Lai 2000), and the recombinant proteins were further resolved by SDS-PAGE, recovered, and renatured by dialysis as described (Yamaguchi et al. 1999b).
GST pull-down assays
GST pull-down assays were carried out as described (Yamaguchi et al. 2001). Briefly, 1 µg of various GST-HDAg derivatives were coupled to glutathione Sepharose beads (GE Healthcare) and then incubated with 100 µL (~600 µg) of HeLa nuclear extract for 1 h at 4 °C. After extensive washing with 0.1HGKEDN [20 mM HEPES (pH 7.9), 20% glycerol, 0.1 M KCl, 0.4 mM EDTA, 0.1% NP-40], bound materials were eluted with 2% SDS.
Photocrosslinking and proteolytic mapping
First, Cys substitution mutants of His-HDAg (20 nmol) were fully reduced with 146 mM 2ME, and the reducing agent was removed by gel filtration using 25 mM Tris (pH 7.9), 400 mM NaCl and 1 mM EDTA as column buffer. All the subsequent steps prior to photocrosslinking were carried out in dim light. AET (12.5 nmol, Invitrogen) was radioiodinated with 2 mCi (1 nmol) of [125I] NaI in a IODO-GEN tube (Pierce) and then introduced into one of the HDAg Cys derivatives (~1 mL, 12.5 nmol) by incubation for 30 min at 25 °C. After removal of unreacted AET by gel filtration, aliquots (10 pmol) of the HDAg AET derivatives were incubated with 50 µL (~300 µg) of HeLa nuclear pellet extract (Hasegawa et al. 2003) for 2 h at 4 °C and then irradiated with 365 nm UV light (0.5 J/cm2) at 25 °C. Unless otherwise stated, the AET moiety was cleaved off from HDAg by incubation with 146 mM 2ME for 15 min at 37 °C. Pol II was immunoprecipitated with anti-Rpb1 antibody (Covance, 8WG16) and protein G Sepharose (GE Healthcare).
For proteolytic mapping, gel slices containing radioactive Rpb1 and Rpb2 were crushed into fine pieces and soaked in 1 mM EDTA, 0.1% SDS, and 1 mM DTT for 16 h. Supernatants were lyophilized, resuspended in 2 M hydroxylamine and 200 mM K2CO3 (pH 10), and incubated for the indicated times. Digested protein samples were then concentrated by trichloroacetic acid precipitation and resolved by SDS-PAGE. Detection was carried out by silver staining, autoradiography, and immunoblotting with antibodies against the extreme N-terminus of Rpb1 (Santa Cruz, N-20) and the extreme C-terminus of Rpb2. The latter antibody was prepared by immunization of a rabbit with the synthetic peptide SRDGGLRFGEMERDC, which corresponds to amino acids 1079–1093 of Rpb2.
In vitro transcription assays
FH-Pol II was purified from HeLa FH3 cells, HeLa derivative cells that constitutively express Flag- and His-tagged human Rpb3, by single-step anti-Flag immunoaffinity chromatography as described (Hasegawa et al. 2003). Transcription with an oligo-dC-tailed template was carried out as described (Yamaguchi et al. 1999a). Transcription with reconstituted elongation complexes was carried out essentially as described (Sidorenkov et al. 1998; Kireeva et al. 2000; Komissarova et al. 2003). First, the oligonucleotide RNA9 was end-labeled with 32P and annealed to the template DNA strand oligonucleotide TDS50. Then, 0.75 pmol of the DNA/RNA duplex was incubated with 20 ng of purified FH-Pol II for 10 min at 30 °C. After an additional 10 min incubation with 60 pmol of the non-template DNA strand oligonucleotide NDS50, elongation complexes were isolated with Ni2+-NTA agarose beads and washed extensively. Then, all four NTPs were added, elongation was allowed to proceed for the indicated times, and transcripts were directly analyzed on 8 M urea-20% polyacrylamide gels. Radioactive signals were quantified using a Storm 860 imager (Molecular Dynamics) and expressed as arbitrary units. For example, the rate of RNA10 synthesis was determined from band intensities of RNA10 plus all the longer transcripts.
For RNA sequence analysis, transcripts were amplified by RT-PCR, PCR products were cloned into pBluescript SK+, and a number of independent clones were subjected to dideoxy sequencing. To avoid amplification of DNA templates at the step of RT-PCR, RNA19 (5'-CAGCAGUUAGAUCGAGAGG-3'), carrying a 10-nt 5' non-complementary tail, was used instead of RNA9 to reconstitute elongation complexes, and resulting transcripts were amplified with the primers 5'-CAGCAGTTAGATCGAGA-3' and 5'-GGTGTAGCTTGGGTTG-3'. PCR cycles were determined for each case so that amplification of DNA templates was negligible.
Structural analysis in silico
The MOE 2004.03 software was used for molecular modeling and energy calculations. The crystal structure of a yeast Pol II transcribing complex in a post-translocation state (Protein Data Bank ID, 1SFO [PDB] ; Westover et al. 2004) was used as a starting structure to construct structural models bearing G or 8-oxoG in an anti or syn conformation at position i + 1 in the template strand. The four structures, G(anti), G(syn), 8-oxoG(anti) and 8-oxoG(syn), were subjected to a constrained energy minimization using the AMBER 94 force field, and potential energy was calculated for each resulting structure. The potential energy of G(syn) relative to that of G(anti) was 5.162 kcal/mol, whereas the potential energy of 8-oxoG(syn) relative to that of 8-oxoG(anti) was –14.188 kcal/mol, suggesting that the syn conformation is energetically more favorable than the anti conformation in the elongation complex.
To give insights into the destabilizing effect of the C8 oxygen of 8-oxoG in the anti conformation, we compared the structural models of 8-oxoG(anti) and G(anti). Hsu et al. (2004) suggested that the bacterial DNA Pol I ternary complex carrying 8-oxoG in the anti conformation is relatively unstable because of steric hindrance between the C8 oxygen of 8-oxoG and the adjacent sugar and phosphate moieties. To test whether this also applies to the Pol II elongation complex, distances between the C8 oxygen of 8-oxoG and oxygens of the DNA backbone were measured in our models. For the 8-oxoG(anti) model, the distances of O4', O5', and O1P from the C8 oxygen were 3.20, 2.52 and 3.96 Å, respectively, whereas corresponding distances for the G(anti) model were 3.17, 2.12 and 3.78 Å, respectively. Thus, there was a small but significant increase of the distances in the 8-oxoG(anti) model, suggesting that 8-oxoG in the anti conformation, which can pair with cytosine, causes template distortion.
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Acknowledgements |
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Footnotes |
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1Present address: University of Tübingen, Tübingen, Germany.
2Present address: Active Motif, Shinjuku, Tokyo, Japan.
* Correspondence: E-mail: hhanda{at}bio.titech.ac.jp |
References |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Bushnell, D.A., Westover, K.D., Davis, R.E. & Kornberg, R.D. (2004) Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Angstroms. Science 303, 983–988.
Chang, J., Nie, X., Gudima, S. & Taylor, J. (2006) Action of inhibitors on accumulation of processed hepatitis delta virus RNAs. J. Virol. 80, 3205–3214.
Chao, M., Hsieh, S.-Y. & Taylor, J. (1990) Role of two forms of the hepatitis delta virus antigen: evidence for a mechanism of self-limiting genome replication. J. Virol. 64, 5066–5069.
Chen, H.T. & Hahn, S. (2003) Mapping the binding site for the TFIIB zinc ribbon domain within the preinitiation complex. Mol. Cell 12, 437–447.[CrossRef][Medline]
Chen, H.T. & Hahn, S. (2004) Mapping the location of TFIIB within the RNA polymerase II transcription preinitiation complex: a model for the structure of the PIC. Cell 119, 169–180.[CrossRef][Medline]
Chen, Y., Ebright, Y.W. & Ebright, R.H. (1994) Identification of the target of a transcription activator protein by protein–protein photocrosslinking. Science 265, 90–92.
Chung, W.H., Craighead, J.L., Chang, W.H., Ezeokonkwo, C., Bareket-Samish, A., Kornberg, R.D. & Asturias, F.J. (2003) RNA polymerase II/TFIIF structure and conserved organization of the initiation complex. Mol. Cell 12, 1003–1013.[CrossRef][Medline]
Filipovska, J. & Konarska, M.M. (2000) Specific HDV RNA-templated transcription by pol II in vitro. RNA 6, 41–54.[Abstract]
Foster, J.E., Holmes, S.F. & Erie, D.A. (2001) Allosteric binding of nucleoside triphosphates to RNA polymerase regulates transcription elongation. Cell 106, 243–252.[CrossRef][Medline]
Gong, X.Q., Zhang, C., Feig, M. & Burton, Z.F. (2005) Dynamic error correction and regulation of downstream bubble opening by human RNA polymerase II. Mol. Cell 18, 461–470.[CrossRef][Medline]
Greco-Stewart, V.S., Miron, P., Abrahem, A. & Pelchat, M. (2007) The human RNA polymerase II interacts with the terminal stem-loop regions of the hepatitis delta virus RNA genome. Virology 357, 68–78.[CrossRef][Medline]
Hartzog, G.A. (2003) Transcription elongation by RNA polymerase II. Curr. Opin. Genet. Dev. 13, 119–126.[CrossRef][Medline]
Hasegawa, J., Endou, M., Narita, T., Yamada, T., Yamaguchi, Y., Wada, T. & Handa, H. (2003) A rapid purification method for human RNA polymerase II by two-step affinity chromatography. J. Biochem. 133, 133–138.
Hsu, G.W., Ober, M., Carell, T. & Beese, L.S. (2004) Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase. Nature 431, 217–221.[CrossRef][Medline]
Huang, Y., Beaudry, A., McSwiggen, J. & Sousa, R. (1997) Determinants of ribose specificity in RNA polymerization: effects of Mn2+ and deoxynucleoside monophosphate incorporation into transcripts. Biochemistry 36, 13718–13728.[CrossRef][Medline]
Jeon, C. & Agarwal, K. (1996) Fidelity of RNA polymerase II transcription controlled by elongation factor TFIIS. Proc. Natl. Acad. Sci. USA 93, 13677–13682.
Kettenberger, H., Armache, K.J. & Cramer, P. (2003) Architecture of the RNA polymerase II-TFIIS complex and implications for mRNA cleavage. Cell 114, 347–357.[CrossRef][Medline]
Kettenberger, H., Armache, K.J. & Cramer, P. (2004) Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. Mol. Cell 16, 955–965.[CrossRef][Medline]
Kireeva, M.L., Komissarova, N., Waugh, D.S. & Kashlev, M. (2000) The 8-nucleotide-long RNA : DNA hybrid is a primary stability determinant of the RNA polymerase II elongation complex. J. Biol. Chem. 275, 6530–6536.
Komissarova, N., Kireeva, M.L., Becker, J., Sidorenkov, I. & Kashlev, M. (2003) Engineering of elongation complexes of bacterial and yeast RNA polymerases. Methods Enzymol. 371, 233–251.[Medline]
Kuraoka, I., Endou, M., Yamaguchi, Y., Wada, T., Handa, H. & Tanaka, K. (2003) Effects of endogeneous DNA base lesions on transcription elongation by mammalian RNA polymerase II: implications for transcription-coupled DNA repair and transcriptional mutagenesis. J. Biol. Chem. 278, 7294–7299.
Lai, M.M. (2005) RNA replication without RNA-dependent RNA polymerase: surprises from hepatitis delta virus. J. Virol. 79, 7951–7958.
Moraleda, G. & Taylor, J. (2001) Host RNA polymerase requirements for transcription of the human hepatitis delta virus genome. J. Virol. 75, 10161–10169.
Narita, T., Yamaguchi, Y., Yano, K., Sugimoto, S., Chanarat, S., Wada, T., Kim, D., Hasegawa, J., Omori, M., Inukai, N., Endoh, M., Yamada, T. & Handa, H. (2003) Human transcription elongation factor NELF: identification of novel subunits and reconstitution of the functionally active complex. Mol. Cell. Biol. 23, 1863–1873.
Nedialkov, Y.A., Gong, X.Q., Hovde, S.L., Yamaguchi, Y., Handa, H., Geiger, J.H., Yan, H. & Burton, Z.F. (2003) NTP-driven translocation by human RNA polymerase II. J. Biol. Chem. 278, 18303–18312.
Niu, W., Kim, Y., Tau, G., Heyduk, T. & Ebright, R.H. (1996) Transcription activation at class II CAP-dependent promoters: two interactions between CAP and RNA polymerase. Cell 87, 1123–1134.[CrossRef][Medline]
Renner, D.B., Yamaguchi, Y., Wada, T., Handa, H. & Price, D.H. (2001) A highly purified RNA polymerase II elongation control system. J. Biol. Chem. 276, 42601–42609.
Sheu, G.T. & Lai, M.M. (2000) Recombinant hepatitis delta antigen from E. coli promotes hepatitis delta virus RNA replication only from the genomic strand but not the antigenomic strand. Virology 278, 578–586.[CrossRef][Medline]
Shibutani, S., Takeshita, M. & Grollman, A.P. (1991) Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature 349, 431–434.[CrossRef][Medline]
Shilatifard, A., Conaway, R.C. & Conaway, J.W. (2003) The RNA polymerase II elongation complex. Annu. Rev. Biochem. 72, 693–715.[CrossRef][Medline]
Sidorenkov, I., Komissarova, N. & Kashlev, M. (1998) Crucial role of the RNA : DNA hybrid in the processivity of transcription. Mol. Cell 2, 55–64.[CrossRef][Medline]
Sims, R.J. 3rd, Belotserkovskaya, R. & Reinberg, D. (2004) Elongation by RNA polymerase II: the short and long of it. Genes Dev. 18, 2437–2468.
Taylor, J.M. (2006) Structure and replication of hepatitis delta virus RNA. Curr. Top. Microbiol. Immunol. 307, 1–23.[Medline]
Thomas, M.J., Platas, A.A. & Hawley, D.K. (1998) Transcriptional fidelity and proofreading by RNA polymerase II. Cell 93, 627–637.[CrossRef][Medline]
Wada, T., Takagi, T., Yamaguchi, Y., Ferdous, A., Imai, T., Hirose, S., Sugimoto, S., Yano, K., Hartzog, G.A., Winston, F., Buratowski, S. & Handa, H. (1998a) DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. Genes Dev. 12, 343–356.
Wada, T., Takagi, T., Yamaguchi, Y., Watanabe, D. & Handa, H. (1998b) Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro. EMBO J. 17, 7395–7403.[CrossRef][Medline]
Westover, K.D., Bushnell, D.A. & Kornberg, R.D. (2004) Structural basis of transcription: separation of RNA from DNA by RNA polymerase II. Science 303, 1014–1016.
Yamada, T., Yamaguchi, Y., Inukai, N., Kamijo, S., Mura, T. & Handa, H. (2006) P-TEFb-mediated phosphorylation of the hSpt5 C-terminal repeats is critical to processive transcription elongation. Mol. Cell 21, 227–237.[CrossRef][Medline]
Yamaguchi, Y., Deléhouzée, S. & Handa, H. (2002) HIV and HDV: evolution takes different paths to relieve blocks in transcriptional elongation. Microbes Infect. 4, 1169–1175.[CrossRef][Medline]
Yamaguchi, Y., Filipovska, J., Yano, K., Furuya, A., Inukai, N., Narita, T., Wada, T., Sugimoto, S., Konarska, M.M. & Handa, H. (2001) Stimulation of RNA polymerase II elongation by hepatitis delta antigen. Science 293, 124–127.
Yamaguchi, Y., Takagi, T., Wada, T., Yano, K., Furuya, A., Sugimoto, S., Hasegawa, J. & Handa, H. (1999a) NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell 97, 41–51.[CrossRef][Medline]
Yamaguchi, Y., Wada, T., Watanabe, D., Takagi, T., Hasegawa, J. & Handa, H. (1999b) Structure and function of the human transcription elongation factor DSIF. J. Biol. Chem. 274, 8085–8092.
Zhang, C., Yan, H. & Burton, Z.F. (2003) Combinatorial control of human RNA polymerase II (RNAP II) pausing and transcript cleavage by transcription factor IIF, hepatitis delta antigen, and stimulatory factor II. J. Biol. Chem. 278, 50101–50111.
Zuccola, H.J., Rozzelle, J.E., Lemon, S.M., Erickson, B.W. & Hogle, J.M. (1998) Structural basis of the oligomerization of hepatitis delta antigen. Structure 6, 821–830.[Medline]
Accepted: 10 April 2007
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-32P] UTP. (C) Elongation complexes were reconstituted with TDS50 or TDS50(8oG), 32P-labeled RNA9, NDS50 and FH-Pol II, and then transcription was allowed to proceed for various times at the indicated concentrations of NTPs. Where indicated, a saturating amount of HDAg was included. The synthesis rates of RNA10 (10+), RNA23 (23+), and the full-length transcript (FL) are shown in arbitrary units below. (D) Transcripts from the TDS50(8oG) template were amplified and cloned, and a number of independent clones were sequenced, as described in Experimental procedures. Actual numbers of the sequences found to contain the indicated nucleotides at position +24 are shown in pie charts. Possible effects of NTP concentrations, HDAg, Mn2+, and TFIIS on the incorporation of ATP and CTP were analyzed by Fisher's exact probability test, and two-sided P values are indicated below.