- Email: [email protected]
The primase active site is on the outside of the hexameric bacteriophage T7 gene 4 helicase-primase ring1
doi:10.1006/jmbi.2001.4932 available online at http://www.idealibrary.com on
J. Mol. Biol. (2001) 311, 951±956
COMMUNICATION
The Primase Active Site is on the Outside of the Hexameric Bacteriophage T7 Gene 4 HelicasePrimase Ring Margaret S. VanLoock1{, Yen-Ju Chen1{, Xiong Yu1, Smita S. Patel2 and Edward H. Egelman1* 1
Department of Biochemistry and Molecular Genetics University of Virginia Health Sciences, Box 800733 Charlottesville, VA 229080733, USA 2
Department of Biochemistry Robert Wood Johnson Medical Center, Piscataway, NJ 088545635, USA
Gene 4 of bacteriophage T7 encodes a protein (gp4) that can translocate along single-stranded DNA, couple the unwinding of duplex DNA with the hydrolysis of dTTP, and catalyze the synthesis of short RNA oligoribonucleotides for use as primers by T7 DNA polymerase. Electron microscopic studies have shown that gp4 forms hexameric rings, and X-ray crystal structures of the gp4 helicase domain and of the highly homologous RNA polymerase domain of Escherichia coli DnaG have been determined. Earlier biochemical studies have shown that when singlestranded DNA is bound to the hexameric ring, the primase domain remains accessible to free DNA. Given these results, a model was suggested in which the primase active site in the gp4 hexamer is located on the outside of the hexameric ring. We have used electron microscopy and single-particle image analysis to examine T7 gp4, and have determined that the primase active site is located on the outside of the hexameric ring, and therefore provide direct structural support for this model. # 2001 Academic Press
*Corresponding author
Keywords: protein-DNA complexes; ring proteins; electron microscopy; image analysis; molecular modeling
Gene 4 of bacteriophage T7 encodes two proteins, gp4a (63 kDa) and gp4b (56 kDa), which are translated from separate in-frame start sites1 ± 4 (Figure 1(a)). Electron microscopy (EM) studies have shown that both gp4a and gp4b form hexameric rings5 ± 7 with two distinct domains and a large central cavity that is the binding site for single-stranded DNA (ssDNA).8 ± 10 Both proteins can translocate along ssDNA at the replication fork junction in a 50 -30 direction, and couple the unwinding of duplex DNA with the hydrolysis of dTTP.3,11,12 In addition, gp4a can also catalyze the template-directed synthesis of short RNA oligoribonucleotides2,13,14 for use as primers by T7 DNA polymerase.15,16 In contrast, gp4b can only catalyze the synthesis of random diribonucleotides because it lacks the N-terminal 63 residues that contain a {These authors contributed equally to this work. Abbreviations used: EM, electron microscopy; ssDNA, single-stranded DNA. E-mail address of the corresponding author: [email protected] 0022-2836/01/050951±6 $35.00/0
Cys4 zinc ®nger motif (Figure 1(a)) necessary for sequence-speci®c interactions with primase recognition sites on DNA.3,17 X-ray crystal structures of the gp4 helicase domain (residues 262-549;18,19 Figure 1(a)) and of the highly homologous RNA polymerase domain of Escherichia coli DnaG have been determined (Figure 1(a) and (b)).20 The location of the active site in the DnaG RNA polymerase has also been identi®ed (Figure 1(b));20 however, the relative orientation of this region after binding to the DnaB hexameric helicase was not determined. In the E. coli DnaB-DnaG complex there is a 6:1 stoichiometry of helicase and primase proteins;20 in Bacillus stearothermophilus the ratio is 6:2 or 6:3.21 For T7 gp4 the stoichiometry is 6:6, since both the helicase and primase domains are contained within the same protein (Figure 1(a)). Several models for the location of the primase active sites have been suggested. In T7 gp4 the primase active site was proposed to face the outside of the ring,22 while in the E. coli DnaB-DnaG complex two locations for the primase active site have been suggested, one # 2001 Academic Press
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Primase Active Site of T7 gp4 Hexamer
Figure 1. (a) Domain organization of the T7 gp4a and gp4b proteins. The primase domain is located at the N terminus of the protein followed by the linker region and the helicase domain. The highlighted and numbered boxes represent the six highly conserved primase motifs.31 The N-terminal Zn2 domain and the residues included in the DnaG20 and T7 helicase18,19 X-ray crystals structures have been indicated. (b) Sequence alignment of the E. coli DnaG (black) and T7 gp4 primases (purple).31 The six highly conserved motifs (gray boxes) and the residues proposed to be involved in the active site (red) are highlighted.20
on the outside of the ring, and the other on the inside, with the latter being more favored.20 We have used EM and single-particle image analysis to examine the T7 gp4a and gp4b proteins, and have determined the location of the primase active site in the hexameric ring. EM reconstruction Three-dimensional reconstructions of negatively stained gp4a and gp4b were generated using 26,000 images for each of the proteins with bound dTMP-PCP (Figure 2(a)). The resulting reconstructions are greatly improved over the previously reported gp4b EM reconstruction,9 and Ê ). In show signi®cantly higher resolution (18 A these reconstructions, both gp4a and gp4b form a hexameric ring with two distinct domains and an open, central channel that forms the binding site for DNA8 ± 10 (Figures 3(a) and (b) and 4(d)). Given the improved resolution and the availability of X-ray crystal structures of the T7 helicase domain18,19 and a homologous primase domain,20 we have been able to clearly identify both the primase and helicase domains in the gp4 hexameric ring (Figure 3(c)). In the EM reconstruction, the helicase domain is strongly handed (Figure 3(a) and (b)), a feature observed in both the helicase crystal structure (Figure 4(a) and (e)) and in many
other ring helicases.23,24 The primase domain forms a slightly narrower ring than the helicase domain and adopts a sharply curved cashew shape (Figure 3). This domain is connected to the helicase through a continuous piece of density that can be attributed to the linker domain (Figures 1(a) and 3(c)). The outside diameter of the gp4 hexamer is Ê and the diameter of the central channel 130 A Ê. measures 30 A The 63 residue N-terminal zinc-binding domain is not clearly resolved in the gp4a reconstruction, suggesting that it may be highly ¯exible or disordered. In fact, using 50 % of the gp4a images, we independently generated a three-dimensional reconstruction (Figure 3(a)) that is nearly identical with that for gp4b (Figure 3(b)), which has no zinc domain. This reconstruction is extremely stable in that the ®nal reconstruction is independent of the starting model. However, reconstructions generated from the other 50 % of the gp4a images yielded a variety of three-dimensional reconstructions, suggesting that these images represent particles in a continuum of conformational states. Reconstructions generated using this set of images all had slightly larger primase domains, and showed signi®cant variations in the linker region (data not shown). The instability associated with these three-dimensional reconstructions may result from multiple conformations and/or disorder in
953
Primase Active Site of T7 gp4 Hexamer
Figure 2. (a) Electron micrograph of the T7 gp4b hexÊ ). The americ protein (the scale bar represents 1000 A insets are reference-free averages of (b) 735 and (c) 828 images of gp4a and gp4b, respectively (the scale bar Ê ). The images were selected by using represents 100 A multi-reference alignment against different projections of the ®nal three-dimensional reconstruction, and choosing those images that had the highest cross-correlations against projections of the reference volume corresponding to tilts of 10 or less of the symmetry axis from the normal to the plane of projection. No symmetry has been imposed in generating these averages. In both proteins, there are six subunits interacting to form a strongly handed hexameric ring. The T7 gp4a0 and gp4b proteins were prepared as described.33 gp4a0 is identical in function and sequence with gp4a except for a Met62Leu mutation.33 This mutation allows for the expression of gp4a0 alone, since the Met64 codon is the initiation site used for expression of gp4b. Specimens were applied to glow discharged grids, stained with 2 % (w/v) uranyl acetate, and imaged under minimal dose conditions in a JEOL 1200 EXII electron microscope, at nominal magni®cation of 30,000.
both the linker region and zinc domain. The fact that most of these differences appear in the linker region is consistent with the suggestion that the zinc domain interacts simultaneously with both the helicase and primase domains.25 We cannot distinguish between multiple conformations of a mobile domain and complete unfolding to explain the inability to clearly visualize the Zn2 domain in gp4a after extensive averaging of thousands of images and the imposition of 6-fold symmetry on the reconstruction. In another helicase, Rep, a domain containing 165 residues is seen in two different conformations related by a 130 rotation in two subunits in the crystal structure.26 In another multimer-forming protein, bacterial ¯agellin, the 65 N-terminal residues are unfolded in solution.27 Similarly, the N-terminal Zn2 domain in gp4a may also be disordered when not bound to its cognate recognition sequence.
In creating the gp4a and gp4b three-dimensional reconstructions, we imposed C6 symmetry, although we cannot exclude the possibility that pseudo-C6 symmetry exists with true C2 or C3 symmetries. Other proteins that form hexameric rings such as RecA,28 E. coli replication helicase DnaB,29 papilloma virus replication helicase E1,30 and bacteriophage SPP1 replication helicase gp4023 are known to exist in two quaternary states, one with C6 symmetry and the other with C3, the latter resulting from a trimer of dimers. However, after extensive searching for alternative symmetries in the gp4a and gp4b images, we were unable to ®nd a signi®cant number of images consistent with structures having a symmetry other than C6. This is in agreement with the previously published gp4b EM reconstruction in which no 2 or 3-fold rotational component was found.6 Locating the primase active site According to sequence alignments of the gp4 primase domain and residues contained in the DnaG crystal structure, approximately 192 residues are highly homologous, including ®ve distinct regions conserved among homologous bacterial and bacteriophage primases (the sixth conserved region, which includes the zinc domain, was not contained in the crystal structure)31 and the proposed active site20 (Figure 1(b)). We positioned the crystal structure of the gp4 helicase18 and the conserved region from the DnaG RNA polymerase domain structure20 into the gp4a (Figure 3(c)) and gp4b three-dimensional reconstructions using manual rigid-body rotation methods. The 6-fold symmetry was then imposed on the ®tted structures to generate a model for the hexameric rings. The resulting models for both the gp4a and gp4b reconstructions were very similar with a 20 rotation of the primase structure when the helicase domains are aligned, and small variations in the linker region (Figure 3(a) and (b)). In the crystal structure of the T7 helicase domain18 there are three different subunit orientations (``A'', ``B'' and ``C'') with a 2-fold crystallographic symmetry generating the other three subunits within the ring. This 2-fold symmetry is not striking at low resolution, as shown by an Ê resolution rendered surface of the six sub18 A units in the helicase crystal structure (Figure 4(a)). We have also imposed 6-fold symmetry on this density map (Figure 4(b)). The resulting maps show that the differences between the crystal structure and the EM reconstruction are not due to the imposition of 6-fold symmetry on a 2-fold symmetric structure (Figure 4(a)-(d)) but are the result of either conformational changes in the helicase domain, or a different relative orientation of the subunits. To test this idea and to generate a model for gp4, we ®rst determined which of the three crystallographic subunit orientations provided the best match with the helicase domain in the EM reconstruction. This was accomplished by docking
954
Figure 3. Three-dimensional EM reconstructions of gp4a and gp4b. Side view of the (a) gp4a and (b) gp4b three-dimensional reconstructions. When the helicase domains are aligned, the primase domain is rotated 20 about the 6-fold symmetry axis when gp4b is compared to gp4a. (c) The gp4 helicase18 and DnaG RNA polymerase domain20 X-ray crystal structures have been ®t into the gp4a three-dimensional reconstruction using the crystallography package O.34 The residues constituting the proposed primase active site20 (red) have been highlighted. Both gp4a and gp4b can translocate along ssDNA in a 50 -30 direction and couple the unwinding of duplex DNA with the hydrolysis of dTTP.3,11,12 The direction of gp4 translocation has been indicated (arrow) using a schematic representation of DNA that passes through the central channel of the hexameric ring.8 ± 10 All surfaces are shown at 100 % of the predicted molecular volume, assuming a partial speci®c volume of protein of 0.75 cm3/g. In generating the three-dimensional reconstructions, the negatives were Ê/ densitometered using the Leaf45 scanner (at 3.9 A pixel) and all image analysis employed the SPIDER software package.35 The reconstructions were started by using the existing, lower resolution reconstruction of gp4b9 as an initial model, and then iteratively using the projections of the reconstruction to align images, determine the corresponding Euler angles of the projections, and generate a new reconstruction. This process was repeated 40 times, and proved to be stable, so that there were no signi®cant changes with additional cycles. The gp4a images were separated into two groups, gp4aI and gp4a-II. The gp4a-I set of images produced a single reconstruction while a variety of reconstructions were generated from the gp4a-II set. To test the stability of the reconstructions generating using the gp4a-I set of images, we used reconstructions generated from the gp4a-II set of images as the starting model. After 20 cycles, the reconstruction was identical with that originally generated using the gp4a-I set of images. Additionally, the stability of the gp4b reconstruction was tested using the stable gp4a reconstruction as the
Primase Active Site of T7 gp4 Hexamer
each crystallographic subunit into the EM map. The resulting ®ts showed that the orientation of the C subunit most closely resembled the helicase domain in the EM reconstructions, although additional rotations were necessary to ®nd the optimum position in the EM map (Figure 4(e)). The differences become obvious when comparing an Ê resolution rendered surface of the C subunit 18 A with 6-fold imposed symmetry (Figure 4(c)) and our reconstruction of T7 gp4 (Figure 4(d)). Even after optimizing the ®t of the helicase subunit into the EM map, two a-helices at the outer edge of the helicase domain penetrated outside the surface envelope (Figure 4(e)). This suggests that the helicase domain either undergoes a signi®cant rearrangement from the crystal structure when the primase domain is included, or that the difference in ring structure is a result of crystallographic conditions and/or crystal packing. In fact, a smaller T7 gp4 helicase domain packs in a crystal as a 61 screw, rather than as a hexameric ring,19 showing that crystal packing of this domain can be polymorphic. The T7 helicase crystal structure18 also included an extended N-terminal a-helix that is part of the linker region (residues 245-272, Figure 1(a)) required for hexamerization.32 In our model, this ahelix from the helicase domain in one subunit extends down toward the primase domain of an adjacent subunit (Figure 3(c)). This suggests that the morphological ``subunit'' in the EM reconstruction may contain a helicase domain from one subunit sitting directly above a primase domain from an adjacent subunit (Figure 3(c)), an idea previously suggested by Sawaya et al.,19 based upon the observed packing of the subunits in their crystal. The idea is also consistent with the hexameric18 T7 helicase crystal structure in that the N-terminal a-helix from one helicase subunit extends out of the core domain and interacts with an adjacent subunit in the hexameric ring. It was suggested that this helix might be an important component of the subunit-subunit contacts necessary to stabilize the hexameric ring.18 Interestingly, in the crystal structure of the T7 helicase hexamer, this N-terminal a-helix interacts with two helices on the outer edge of the adjacent subunit. The ®tting of this structure into the EM map suggests that these two helices must undergo a conformational rearrangement from that seen in the crystal (Figure 3(c)). As a result, if the contacts between these two helices and the N-terminal a-helix are maintained, the Nterminal a-helix must also undergo a conformational change. This suggests that the arrangement of the T7 gp4 morphological subunit in the starting model, and it converged back to the original gp4b reconstruction after about ten cycles. The resolution of the three-dimensional reconstruction was calculated using the criterion of the Fourier shell correlation falling to 0.5 between reconstructions generated from two halves of the data set.
955
Primase Active Site of T7 gp4 Hexamer
tions (Figure 3(c)). This is consistent with earlier biochemical studies showing that in the presence of a non-hydrolyzable analogue of dTTP, gp4 is locked on the ssDNA to which it was initially bound.22 Under these conditions, the primase is able to synthesize primers on a second ssDNA molecule. Given these results, the authors suggested a model in which the primase active site in gp4 is located on the outside of the hexameric ring.22 Our results provide direct structural support for this model.
Acknowledgments This research was supported by NIH GM35269 (to E.H.E.).
References
Ê resolution) of the Figure 4. (a) Rendered surface (18 A gp4 helicase domain X-ray crystal structure,18 and this same density with imposed 6-fold symmetry (b). In the crystal structure of the T7 helicase domain18 there are three unique subunit orientations (``A'', ``B'' and ``C''). The C subunit most closely resembles the orientation of the helicase subunits in the EM reconstruction. This is Ê resolution renobvious when comparing (c) an 18 A dered surface of the C subunit with 6-fold imposed symmetry and (d) the T7 gp4a reconstruction viewed from the top of the helicase domain. (e) Fitting of the helicase structure (green) into the EM reconstruction (grey). The arrow indicates the loop region (red) that connects the N-terminal a-helix (red) of one helicase domain that interacts with an adjacent primase domain.
EM reconstructions may be different from that suggested in the crystal. Alternatively, the N-terminal helix in the helicase domain may adopt a different conformation from that seen in the crystal structure when the primase domain is expressed, therefore altering the relative orientation of the helicase and primase domains. In the absence of the ability to trace the polypeptide chain in the EM reconstruction this will remain ambiguous. The crystal structure of the RNA polymerase domain of DnaG has a very pronounced curvature that forms an electro-positive cleft for binding DNA.20 This curvature is also apparent in the cashew-shaped primase domains in both the gp4a and gp4b reconstructions (Figure 3). It is this curvature that guided the unique placement of the crystal structure into the EM map (Figure 3(c)). Interestingly, the resulting ®t of the DnaG RNA polymerase domain showed that the active site and nucleic acid binding region identi®ed in the DnaG structure20 is facing the outside of the hexameric ring in both the gp4a and gp4b reconstruc-
1. Dunn, J. J. & Studier, F. W. (1983). Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166, 477-535. 2. Bernstein, J. A. & Richardson, C. C. (1989). Characterization of the helicase and primase activities of the 63-kDa component of the bacteriophage T7 gene 4 protein. J. Biol. Chem. 264, 13066-13073. 3. Bernstein, J. A. & Richardson, C. C. (1988). Puri®cation of the 56-kDa component of the bacteriophage T7 primase/helicase and characterization of its nucleoside 50 -triphosphatase activity. J. Biol. Chem. 263, 14891-14899. 4. Mendelman, L. V. & Richardson, C. C. (1991). Requirements for primer synthesis by bacteriophage T7 63-kDa gene 4 protein. Roles of template sequence and T7 56-kDa gene 4 protein. J. Biol. Chem. 266, 23240-23250. 5. Patel, S. S. & Hingorani, M. M. (1993). Oligomeric structure of bacteriophage T7 DNA primase/helicase proteins. J. Biol. Chem. 268, 10668-10675. 6. Egelman, H. H., Yu, X., Wild, R., Hingorani, M. M. & Patel, S. S. (1995). Bacteriophage T7 helicase/primase proteins form rings around single- stranded DNA that suggest a general structure for hexameric helicases. Proc. Natl Acad. Sci. USA, 92, 3869-3873. 7. Notarnicola, S. M., Park, K., Grif®th, J. D. & Richardson, C. C. (1995). A domain of the gene 4 helicase/primase of bacteriophage T7 required for the formation of an active hexamer. J. Biol. Chem. 270, 20215-20224. 8. Ahnert, P. & Patel, S. S. (1997). Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 50 - and 30 -tails of the forked DNA substrate. J. Biol. Chem. 272, 32267-32273. 9. Yu, X., Hingorani, M. M., Patel, S. S. & Egelman, E. H. (1996). DNA is bound within the central hole to one or two of the six subunits of the T7 DNA helicase. Natature Struct. Biol. 3, 740-743. 10. Hacker, K. J. & Johnson, K. A. (1997). A hexameric helicase encircles one DNA strand and excludes the other during DNA unwinding. Biochemistry, 36, 14080-14087. 11. Kolodner, R. & Richardson, C. C. (1977). Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydroly-
956
12.
13.
14.
15.
16.
17.
18.
19.
20. 21.
22.
23.
Primase Active Site of T7 gp4 Hexamer
sis of nucleoside 50 -triphosphates. Proc. Natl Acad. Sci. USA, 74, 1525-1529. Matson, S. W., Tabor, S. & Richardson, C. C. (1983). The gene 4 protein of bacteriophage T7. Characterization of helicase activity. J. Biol. Chem. 258, 1401714024. Matson, S. W. & Richardson, C. C. (1983). DNAdependent nucleoside 50 -triphosphatase activity of the gene 4 protein of bacteriophage T7. J. Biol. Chem. 258, 14009-14016. Tabor, S. & Richardson, C. C. (1981). Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7. Proc. Natl Acad. Sci. USA, 78, 205-209. Scherzinger, E., Lanka, E. & Hillenbrand, G. (1977). Role of bacteriophage T7 DNA primase in the initiation of DNA strand synthesis. Nucl. Acids Res. 4, 4151-4163. Nakai, H. & Richardson, C. C. (1986). Interactions of the DNA polymerase and gene 4 protein of bacteriophage T7. Protein-protein and protein-DNA interactions involved in RNA-primed DNA synthesis. J. Biol. Chem. 261, 15208-15216. Bernstein, J. A. & Richardson, C. C. (1988). A 7-kDa region of the bacteriophage T7 gene 4 protein is required for primase but not for helicase activity. Proc. Natl Acad. Sci. USA, 85, 396-400. Singleton, M. R., Sawaya, M. R., Ellenberger, T. & Wigley, D. B. (2000). Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell, 101, 589-600. Sawaya, M. R., Guo, S., Tabor, S., Richardson, C. C. & Ellenberger, T. (1999). Crystal structure of the helicase domain from the replicative helicaseprimase of bacteriophage T7. Cell, 99, 167-77. Keck, J. L., Roche, D. D., Lynch, A. S. & Berger, J. M. (2000). Structure of the RNA polymerase domain of E. coli primase. Science, 287, 2482-2486. Bird, L. E., Pan, H., Soultanas, P. & Wigley, D. B. (2000). Mapping protein-protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus. Biochemistry, 39, 171-182. Kusakabe, T., Baradaran, K., Lee, J. & Richardson, C. C. (1998). Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site. EMBO J. 17, 1542-1552. Barcena, M., Martin, C. S., Weise, F., Ayora, S., Alonso, J. C. & Carazo, J. M. (1998). Polymorphic quaternary organization of the Bacillus subtilis
24.
25. 26.
27. 28. 29.
30.
31.
32.
33.
34.
35.
bacteriophage SPP1 replicative helicase (G40 P). J. Mol. Biol. 283, 809-819. San Martin, M. C., Gruss, C. & Carazo, J. M. (1997). Six molecules of SV40 large T antigen assemble in a propeller-shaped particle around a channel. J. Mol. Biol. 268, 15-20. Kusakabe, T. & Richardson, C. C. (1996). The role of the zinc motif in sequence recognition by DNA primases. J. Biol. Chem. 271, 19563-19570. Korolev, S., Hsieh, J., Gauss, G. H., Lohman, T. M. & Waksman, G. (1997). Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP. Cell, 90, 635-647. Vonderviszt, F., Kanto, S., Aizawa, S. & Namba, K. (1989). Terminal regions of ¯agellin are disordered in solution. J. Mol. Biol. 209, 127-133. Yu, X. & Egelman, E. H. (1997). The RecA hexamer is a structural homologue of ring helicases. Nature Struct. Biol. 4, 101-104. Yu, X., Jezewska, M. J., Bujalowski, W. & Egelman, E. H. (1996). The hexameric E. coli DnaB helicase can exist in different quaternary states. J. Mol. Biol. 259, 7-14. Fouts, E. T., Yu, X., Egelman, E. H. & Botchan, M. R. (1999). Biochemical and electron microscopic image analysis of the hexameric E1 helicase. J. Biol. Chem. 274, 4447-4458. Ilyina, T. V., Gorbalenya, A. E. & Koonin, E. V. (1992). Organization and evolution of bacterial and bacteriophage primase-helicase systems. J. Mol. Evol. 34, 351-357. Guo, S., Tabor, S. & Richardson, C. C. (1999). The linker region between the helicase and primase domains of the bacteriophage T7 gene 4 protein is critical for hexamer formation. J. Biol. Chem. 274, 30303-30309. Patel, S. S., Rosenberg, A. H., Studier, F. W. & Johnson, K. A. (1992). Large scale puri®cation and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation. J. Biol. Chem. 267, 15013-15021. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. (1991). Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallog. sect. A, 47, 110-119. Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M. & Leith, A. (1996). SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related ®elds. J. Struct. Biol. 116, 190-199.
Edited by W. Baumeister (Received 12 April 2001; received in revised form 5 July 2001; accepted 9 July 2001)
J. Mol. Biol. (2001) 311, 951±956
COMMUNICATION
The Primase Active Site is on the Outside of the Hexameric Bacteriophage T7 Gene 4 HelicasePrimase Ring Margaret S. VanLoock1{, Yen-Ju Chen1{, Xiong Yu1, Smita S. Patel2 and Edward H. Egelman1* 1
Department of Biochemistry and Molecular Genetics University of Virginia Health Sciences, Box 800733 Charlottesville, VA 229080733, USA 2
Department of Biochemistry Robert Wood Johnson Medical Center, Piscataway, NJ 088545635, USA
Gene 4 of bacteriophage T7 encodes a protein (gp4) that can translocate along single-stranded DNA, couple the unwinding of duplex DNA with the hydrolysis of dTTP, and catalyze the synthesis of short RNA oligoribonucleotides for use as primers by T7 DNA polymerase. Electron microscopic studies have shown that gp4 forms hexameric rings, and X-ray crystal structures of the gp4 helicase domain and of the highly homologous RNA polymerase domain of Escherichia coli DnaG have been determined. Earlier biochemical studies have shown that when singlestranded DNA is bound to the hexameric ring, the primase domain remains accessible to free DNA. Given these results, a model was suggested in which the primase active site in the gp4 hexamer is located on the outside of the hexameric ring. We have used electron microscopy and single-particle image analysis to examine T7 gp4, and have determined that the primase active site is located on the outside of the hexameric ring, and therefore provide direct structural support for this model. # 2001 Academic Press
*Corresponding author
Keywords: protein-DNA complexes; ring proteins; electron microscopy; image analysis; molecular modeling
Gene 4 of bacteriophage T7 encodes two proteins, gp4a (63 kDa) and gp4b (56 kDa), which are translated from separate in-frame start sites1 ± 4 (Figure 1(a)). Electron microscopy (EM) studies have shown that both gp4a and gp4b form hexameric rings5 ± 7 with two distinct domains and a large central cavity that is the binding site for single-stranded DNA (ssDNA).8 ± 10 Both proteins can translocate along ssDNA at the replication fork junction in a 50 -30 direction, and couple the unwinding of duplex DNA with the hydrolysis of dTTP.3,11,12 In addition, gp4a can also catalyze the template-directed synthesis of short RNA oligoribonucleotides2,13,14 for use as primers by T7 DNA polymerase.15,16 In contrast, gp4b can only catalyze the synthesis of random diribonucleotides because it lacks the N-terminal 63 residues that contain a {These authors contributed equally to this work. Abbreviations used: EM, electron microscopy; ssDNA, single-stranded DNA. E-mail address of the corresponding author: [email protected] 0022-2836/01/050951±6 $35.00/0
Cys4 zinc ®nger motif (Figure 1(a)) necessary for sequence-speci®c interactions with primase recognition sites on DNA.3,17 X-ray crystal structures of the gp4 helicase domain (residues 262-549;18,19 Figure 1(a)) and of the highly homologous RNA polymerase domain of Escherichia coli DnaG have been determined (Figure 1(a) and (b)).20 The location of the active site in the DnaG RNA polymerase has also been identi®ed (Figure 1(b));20 however, the relative orientation of this region after binding to the DnaB hexameric helicase was not determined. In the E. coli DnaB-DnaG complex there is a 6:1 stoichiometry of helicase and primase proteins;20 in Bacillus stearothermophilus the ratio is 6:2 or 6:3.21 For T7 gp4 the stoichiometry is 6:6, since both the helicase and primase domains are contained within the same protein (Figure 1(a)). Several models for the location of the primase active sites have been suggested. In T7 gp4 the primase active site was proposed to face the outside of the ring,22 while in the E. coli DnaB-DnaG complex two locations for the primase active site have been suggested, one # 2001 Academic Press
952
Primase Active Site of T7 gp4 Hexamer
Figure 1. (a) Domain organization of the T7 gp4a and gp4b proteins. The primase domain is located at the N terminus of the protein followed by the linker region and the helicase domain. The highlighted and numbered boxes represent the six highly conserved primase motifs.31 The N-terminal Zn2 domain and the residues included in the DnaG20 and T7 helicase18,19 X-ray crystals structures have been indicated. (b) Sequence alignment of the E. coli DnaG (black) and T7 gp4 primases (purple).31 The six highly conserved motifs (gray boxes) and the residues proposed to be involved in the active site (red) are highlighted.20
on the outside of the ring, and the other on the inside, with the latter being more favored.20 We have used EM and single-particle image analysis to examine the T7 gp4a and gp4b proteins, and have determined the location of the primase active site in the hexameric ring. EM reconstruction Three-dimensional reconstructions of negatively stained gp4a and gp4b were generated using 26,000 images for each of the proteins with bound dTMP-PCP (Figure 2(a)). The resulting reconstructions are greatly improved over the previously reported gp4b EM reconstruction,9 and Ê ). In show signi®cantly higher resolution (18 A these reconstructions, both gp4a and gp4b form a hexameric ring with two distinct domains and an open, central channel that forms the binding site for DNA8 ± 10 (Figures 3(a) and (b) and 4(d)). Given the improved resolution and the availability of X-ray crystal structures of the T7 helicase domain18,19 and a homologous primase domain,20 we have been able to clearly identify both the primase and helicase domains in the gp4 hexameric ring (Figure 3(c)). In the EM reconstruction, the helicase domain is strongly handed (Figure 3(a) and (b)), a feature observed in both the helicase crystal structure (Figure 4(a) and (e)) and in many
other ring helicases.23,24 The primase domain forms a slightly narrower ring than the helicase domain and adopts a sharply curved cashew shape (Figure 3). This domain is connected to the helicase through a continuous piece of density that can be attributed to the linker domain (Figures 1(a) and 3(c)). The outside diameter of the gp4 hexamer is Ê and the diameter of the central channel 130 A Ê. measures 30 A The 63 residue N-terminal zinc-binding domain is not clearly resolved in the gp4a reconstruction, suggesting that it may be highly ¯exible or disordered. In fact, using 50 % of the gp4a images, we independently generated a three-dimensional reconstruction (Figure 3(a)) that is nearly identical with that for gp4b (Figure 3(b)), which has no zinc domain. This reconstruction is extremely stable in that the ®nal reconstruction is independent of the starting model. However, reconstructions generated from the other 50 % of the gp4a images yielded a variety of three-dimensional reconstructions, suggesting that these images represent particles in a continuum of conformational states. Reconstructions generated using this set of images all had slightly larger primase domains, and showed signi®cant variations in the linker region (data not shown). The instability associated with these three-dimensional reconstructions may result from multiple conformations and/or disorder in
953
Primase Active Site of T7 gp4 Hexamer
Figure 2. (a) Electron micrograph of the T7 gp4b hexÊ ). The americ protein (the scale bar represents 1000 A insets are reference-free averages of (b) 735 and (c) 828 images of gp4a and gp4b, respectively (the scale bar Ê ). The images were selected by using represents 100 A multi-reference alignment against different projections of the ®nal three-dimensional reconstruction, and choosing those images that had the highest cross-correlations against projections of the reference volume corresponding to tilts of 10 or less of the symmetry axis from the normal to the plane of projection. No symmetry has been imposed in generating these averages. In both proteins, there are six subunits interacting to form a strongly handed hexameric ring. The T7 gp4a0 and gp4b proteins were prepared as described.33 gp4a0 is identical in function and sequence with gp4a except for a Met62Leu mutation.33 This mutation allows for the expression of gp4a0 alone, since the Met64 codon is the initiation site used for expression of gp4b. Specimens were applied to glow discharged grids, stained with 2 % (w/v) uranyl acetate, and imaged under minimal dose conditions in a JEOL 1200 EXII electron microscope, at nominal magni®cation of 30,000.
both the linker region and zinc domain. The fact that most of these differences appear in the linker region is consistent with the suggestion that the zinc domain interacts simultaneously with both the helicase and primase domains.25 We cannot distinguish between multiple conformations of a mobile domain and complete unfolding to explain the inability to clearly visualize the Zn2 domain in gp4a after extensive averaging of thousands of images and the imposition of 6-fold symmetry on the reconstruction. In another helicase, Rep, a domain containing 165 residues is seen in two different conformations related by a 130 rotation in two subunits in the crystal structure.26 In another multimer-forming protein, bacterial ¯agellin, the 65 N-terminal residues are unfolded in solution.27 Similarly, the N-terminal Zn2 domain in gp4a may also be disordered when not bound to its cognate recognition sequence.
In creating the gp4a and gp4b three-dimensional reconstructions, we imposed C6 symmetry, although we cannot exclude the possibility that pseudo-C6 symmetry exists with true C2 or C3 symmetries. Other proteins that form hexameric rings such as RecA,28 E. coli replication helicase DnaB,29 papilloma virus replication helicase E1,30 and bacteriophage SPP1 replication helicase gp4023 are known to exist in two quaternary states, one with C6 symmetry and the other with C3, the latter resulting from a trimer of dimers. However, after extensive searching for alternative symmetries in the gp4a and gp4b images, we were unable to ®nd a signi®cant number of images consistent with structures having a symmetry other than C6. This is in agreement with the previously published gp4b EM reconstruction in which no 2 or 3-fold rotational component was found.6 Locating the primase active site According to sequence alignments of the gp4 primase domain and residues contained in the DnaG crystal structure, approximately 192 residues are highly homologous, including ®ve distinct regions conserved among homologous bacterial and bacteriophage primases (the sixth conserved region, which includes the zinc domain, was not contained in the crystal structure)31 and the proposed active site20 (Figure 1(b)). We positioned the crystal structure of the gp4 helicase18 and the conserved region from the DnaG RNA polymerase domain structure20 into the gp4a (Figure 3(c)) and gp4b three-dimensional reconstructions using manual rigid-body rotation methods. The 6-fold symmetry was then imposed on the ®tted structures to generate a model for the hexameric rings. The resulting models for both the gp4a and gp4b reconstructions were very similar with a 20 rotation of the primase structure when the helicase domains are aligned, and small variations in the linker region (Figure 3(a) and (b)). In the crystal structure of the T7 helicase domain18 there are three different subunit orientations (``A'', ``B'' and ``C'') with a 2-fold crystallographic symmetry generating the other three subunits within the ring. This 2-fold symmetry is not striking at low resolution, as shown by an Ê resolution rendered surface of the six sub18 A units in the helicase crystal structure (Figure 4(a)). We have also imposed 6-fold symmetry on this density map (Figure 4(b)). The resulting maps show that the differences between the crystal structure and the EM reconstruction are not due to the imposition of 6-fold symmetry on a 2-fold symmetric structure (Figure 4(a)-(d)) but are the result of either conformational changes in the helicase domain, or a different relative orientation of the subunits. To test this idea and to generate a model for gp4, we ®rst determined which of the three crystallographic subunit orientations provided the best match with the helicase domain in the EM reconstruction. This was accomplished by docking
954
Figure 3. Three-dimensional EM reconstructions of gp4a and gp4b. Side view of the (a) gp4a and (b) gp4b three-dimensional reconstructions. When the helicase domains are aligned, the primase domain is rotated 20 about the 6-fold symmetry axis when gp4b is compared to gp4a. (c) The gp4 helicase18 and DnaG RNA polymerase domain20 X-ray crystal structures have been ®t into the gp4a three-dimensional reconstruction using the crystallography package O.34 The residues constituting the proposed primase active site20 (red) have been highlighted. Both gp4a and gp4b can translocate along ssDNA in a 50 -30 direction and couple the unwinding of duplex DNA with the hydrolysis of dTTP.3,11,12 The direction of gp4 translocation has been indicated (arrow) using a schematic representation of DNA that passes through the central channel of the hexameric ring.8 ± 10 All surfaces are shown at 100 % of the predicted molecular volume, assuming a partial speci®c volume of protein of 0.75 cm3/g. In generating the three-dimensional reconstructions, the negatives were Ê/ densitometered using the Leaf45 scanner (at 3.9 A pixel) and all image analysis employed the SPIDER software package.35 The reconstructions were started by using the existing, lower resolution reconstruction of gp4b9 as an initial model, and then iteratively using the projections of the reconstruction to align images, determine the corresponding Euler angles of the projections, and generate a new reconstruction. This process was repeated 40 times, and proved to be stable, so that there were no signi®cant changes with additional cycles. The gp4a images were separated into two groups, gp4aI and gp4a-II. The gp4a-I set of images produced a single reconstruction while a variety of reconstructions were generated from the gp4a-II set. To test the stability of the reconstructions generating using the gp4a-I set of images, we used reconstructions generated from the gp4a-II set of images as the starting model. After 20 cycles, the reconstruction was identical with that originally generated using the gp4a-I set of images. Additionally, the stability of the gp4b reconstruction was tested using the stable gp4a reconstruction as the
Primase Active Site of T7 gp4 Hexamer
each crystallographic subunit into the EM map. The resulting ®ts showed that the orientation of the C subunit most closely resembled the helicase domain in the EM reconstructions, although additional rotations were necessary to ®nd the optimum position in the EM map (Figure 4(e)). The differences become obvious when comparing an Ê resolution rendered surface of the C subunit 18 A with 6-fold imposed symmetry (Figure 4(c)) and our reconstruction of T7 gp4 (Figure 4(d)). Even after optimizing the ®t of the helicase subunit into the EM map, two a-helices at the outer edge of the helicase domain penetrated outside the surface envelope (Figure 4(e)). This suggests that the helicase domain either undergoes a signi®cant rearrangement from the crystal structure when the primase domain is included, or that the difference in ring structure is a result of crystallographic conditions and/or crystal packing. In fact, a smaller T7 gp4 helicase domain packs in a crystal as a 61 screw, rather than as a hexameric ring,19 showing that crystal packing of this domain can be polymorphic. The T7 helicase crystal structure18 also included an extended N-terminal a-helix that is part of the linker region (residues 245-272, Figure 1(a)) required for hexamerization.32 In our model, this ahelix from the helicase domain in one subunit extends down toward the primase domain of an adjacent subunit (Figure 3(c)). This suggests that the morphological ``subunit'' in the EM reconstruction may contain a helicase domain from one subunit sitting directly above a primase domain from an adjacent subunit (Figure 3(c)), an idea previously suggested by Sawaya et al.,19 based upon the observed packing of the subunits in their crystal. The idea is also consistent with the hexameric18 T7 helicase crystal structure in that the N-terminal a-helix from one helicase subunit extends out of the core domain and interacts with an adjacent subunit in the hexameric ring. It was suggested that this helix might be an important component of the subunit-subunit contacts necessary to stabilize the hexameric ring.18 Interestingly, in the crystal structure of the T7 helicase hexamer, this N-terminal a-helix interacts with two helices on the outer edge of the adjacent subunit. The ®tting of this structure into the EM map suggests that these two helices must undergo a conformational rearrangement from that seen in the crystal (Figure 3(c)). As a result, if the contacts between these two helices and the N-terminal a-helix are maintained, the Nterminal a-helix must also undergo a conformational change. This suggests that the arrangement of the T7 gp4 morphological subunit in the starting model, and it converged back to the original gp4b reconstruction after about ten cycles. The resolution of the three-dimensional reconstruction was calculated using the criterion of the Fourier shell correlation falling to 0.5 between reconstructions generated from two halves of the data set.
955
Primase Active Site of T7 gp4 Hexamer
tions (Figure 3(c)). This is consistent with earlier biochemical studies showing that in the presence of a non-hydrolyzable analogue of dTTP, gp4 is locked on the ssDNA to which it was initially bound.22 Under these conditions, the primase is able to synthesize primers on a second ssDNA molecule. Given these results, the authors suggested a model in which the primase active site in gp4 is located on the outside of the hexameric ring.22 Our results provide direct structural support for this model.
Acknowledgments This research was supported by NIH GM35269 (to E.H.E.).
References
Ê resolution) of the Figure 4. (a) Rendered surface (18 A gp4 helicase domain X-ray crystal structure,18 and this same density with imposed 6-fold symmetry (b). In the crystal structure of the T7 helicase domain18 there are three unique subunit orientations (``A'', ``B'' and ``C''). The C subunit most closely resembles the orientation of the helicase subunits in the EM reconstruction. This is Ê resolution renobvious when comparing (c) an 18 A dered surface of the C subunit with 6-fold imposed symmetry and (d) the T7 gp4a reconstruction viewed from the top of the helicase domain. (e) Fitting of the helicase structure (green) into the EM reconstruction (grey). The arrow indicates the loop region (red) that connects the N-terminal a-helix (red) of one helicase domain that interacts with an adjacent primase domain.
EM reconstructions may be different from that suggested in the crystal. Alternatively, the N-terminal helix in the helicase domain may adopt a different conformation from that seen in the crystal structure when the primase domain is expressed, therefore altering the relative orientation of the helicase and primase domains. In the absence of the ability to trace the polypeptide chain in the EM reconstruction this will remain ambiguous. The crystal structure of the RNA polymerase domain of DnaG has a very pronounced curvature that forms an electro-positive cleft for binding DNA.20 This curvature is also apparent in the cashew-shaped primase domains in both the gp4a and gp4b reconstructions (Figure 3). It is this curvature that guided the unique placement of the crystal structure into the EM map (Figure 3(c)). Interestingly, the resulting ®t of the DnaG RNA polymerase domain showed that the active site and nucleic acid binding region identi®ed in the DnaG structure20 is facing the outside of the hexameric ring in both the gp4a and gp4b reconstruc-
1. Dunn, J. J. & Studier, F. W. (1983). Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166, 477-535. 2. Bernstein, J. A. & Richardson, C. C. (1989). Characterization of the helicase and primase activities of the 63-kDa component of the bacteriophage T7 gene 4 protein. J. Biol. Chem. 264, 13066-13073. 3. Bernstein, J. A. & Richardson, C. C. (1988). Puri®cation of the 56-kDa component of the bacteriophage T7 primase/helicase and characterization of its nucleoside 50 -triphosphatase activity. J. Biol. Chem. 263, 14891-14899. 4. Mendelman, L. V. & Richardson, C. C. (1991). Requirements for primer synthesis by bacteriophage T7 63-kDa gene 4 protein. Roles of template sequence and T7 56-kDa gene 4 protein. J. Biol. Chem. 266, 23240-23250. 5. Patel, S. S. & Hingorani, M. M. (1993). Oligomeric structure of bacteriophage T7 DNA primase/helicase proteins. J. Biol. Chem. 268, 10668-10675. 6. Egelman, H. H., Yu, X., Wild, R., Hingorani, M. M. & Patel, S. S. (1995). Bacteriophage T7 helicase/primase proteins form rings around single- stranded DNA that suggest a general structure for hexameric helicases. Proc. Natl Acad. Sci. USA, 92, 3869-3873. 7. Notarnicola, S. M., Park, K., Grif®th, J. D. & Richardson, C. C. (1995). A domain of the gene 4 helicase/primase of bacteriophage T7 required for the formation of an active hexamer. J. Biol. Chem. 270, 20215-20224. 8. Ahnert, P. & Patel, S. S. (1997). Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 50 - and 30 -tails of the forked DNA substrate. J. Biol. Chem. 272, 32267-32273. 9. Yu, X., Hingorani, M. M., Patel, S. S. & Egelman, E. H. (1996). DNA is bound within the central hole to one or two of the six subunits of the T7 DNA helicase. Natature Struct. Biol. 3, 740-743. 10. Hacker, K. J. & Johnson, K. A. (1997). A hexameric helicase encircles one DNA strand and excludes the other during DNA unwinding. Biochemistry, 36, 14080-14087. 11. Kolodner, R. & Richardson, C. C. (1977). Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydroly-
956
12.
13.
14.
15.
16.
17.
18.
19.
20. 21.
22.
23.
Primase Active Site of T7 gp4 Hexamer
sis of nucleoside 50 -triphosphates. Proc. Natl Acad. Sci. USA, 74, 1525-1529. Matson, S. W., Tabor, S. & Richardson, C. C. (1983). The gene 4 protein of bacteriophage T7. Characterization of helicase activity. J. Biol. Chem. 258, 1401714024. Matson, S. W. & Richardson, C. C. (1983). DNAdependent nucleoside 50 -triphosphatase activity of the gene 4 protein of bacteriophage T7. J. Biol. Chem. 258, 14009-14016. Tabor, S. & Richardson, C. C. (1981). Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7. Proc. Natl Acad. Sci. USA, 78, 205-209. Scherzinger, E., Lanka, E. & Hillenbrand, G. (1977). Role of bacteriophage T7 DNA primase in the initiation of DNA strand synthesis. Nucl. Acids Res. 4, 4151-4163. Nakai, H. & Richardson, C. C. (1986). Interactions of the DNA polymerase and gene 4 protein of bacteriophage T7. Protein-protein and protein-DNA interactions involved in RNA-primed DNA synthesis. J. Biol. Chem. 261, 15208-15216. Bernstein, J. A. & Richardson, C. C. (1988). A 7-kDa region of the bacteriophage T7 gene 4 protein is required for primase but not for helicase activity. Proc. Natl Acad. Sci. USA, 85, 396-400. Singleton, M. R., Sawaya, M. R., Ellenberger, T. & Wigley, D. B. (2000). Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell, 101, 589-600. Sawaya, M. R., Guo, S., Tabor, S., Richardson, C. C. & Ellenberger, T. (1999). Crystal structure of the helicase domain from the replicative helicaseprimase of bacteriophage T7. Cell, 99, 167-77. Keck, J. L., Roche, D. D., Lynch, A. S. & Berger, J. M. (2000). Structure of the RNA polymerase domain of E. coli primase. Science, 287, 2482-2486. Bird, L. E., Pan, H., Soultanas, P. & Wigley, D. B. (2000). Mapping protein-protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus. Biochemistry, 39, 171-182. Kusakabe, T., Baradaran, K., Lee, J. & Richardson, C. C. (1998). Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site. EMBO J. 17, 1542-1552. Barcena, M., Martin, C. S., Weise, F., Ayora, S., Alonso, J. C. & Carazo, J. M. (1998). Polymorphic quaternary organization of the Bacillus subtilis
24.
25. 26.
27. 28. 29.
30.
31.
32.
33.
34.
35.
bacteriophage SPP1 replicative helicase (G40 P). J. Mol. Biol. 283, 809-819. San Martin, M. C., Gruss, C. & Carazo, J. M. (1997). Six molecules of SV40 large T antigen assemble in a propeller-shaped particle around a channel. J. Mol. Biol. 268, 15-20. Kusakabe, T. & Richardson, C. C. (1996). The role of the zinc motif in sequence recognition by DNA primases. J. Biol. Chem. 271, 19563-19570. Korolev, S., Hsieh, J., Gauss, G. H., Lohman, T. M. & Waksman, G. (1997). Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP. Cell, 90, 635-647. Vonderviszt, F., Kanto, S., Aizawa, S. & Namba, K. (1989). Terminal regions of ¯agellin are disordered in solution. J. Mol. Biol. 209, 127-133. Yu, X. & Egelman, E. H. (1997). The RecA hexamer is a structural homologue of ring helicases. Nature Struct. Biol. 4, 101-104. Yu, X., Jezewska, M. J., Bujalowski, W. & Egelman, E. H. (1996). The hexameric E. coli DnaB helicase can exist in different quaternary states. J. Mol. Biol. 259, 7-14. Fouts, E. T., Yu, X., Egelman, E. H. & Botchan, M. R. (1999). Biochemical and electron microscopic image analysis of the hexameric E1 helicase. J. Biol. Chem. 274, 4447-4458. Ilyina, T. V., Gorbalenya, A. E. & Koonin, E. V. (1992). Organization and evolution of bacterial and bacteriophage primase-helicase systems. J. Mol. Evol. 34, 351-357. Guo, S., Tabor, S. & Richardson, C. C. (1999). The linker region between the helicase and primase domains of the bacteriophage T7 gene 4 protein is critical for hexamer formation. J. Biol. Chem. 274, 30303-30309. Patel, S. S., Rosenberg, A. H., Studier, F. W. & Johnson, K. A. (1992). Large scale puri®cation and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation. J. Biol. Chem. 267, 15013-15021. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. (1991). Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallog. sect. A, 47, 110-119. Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M. & Leith, A. (1996). SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related ®elds. J. Struct. Biol. 116, 190-199.
Edited by W. Baumeister (Received 12 April 2001; received in revised form 5 July 2001; accepted 9 July 2001)