place the ssDNA-binding mouth of DnaGdistal from DnaB, orienting the active site ofprimase inward, toward the center of the ring,where it is positioned to accept ssDNA as it isextruded from DnaB (Fig. 4, right). Alterna-tively, it is possible that mechanistic differ-ences between 6:6 and 6 :1 helicase-primasesystems lead to different relative orientationsof the primase active sites. The true relativelocations of these domains awaits high-reso-lution study of the primase-helicase complex-es in E. coli and phage T7.References and Notes1. D. Brutlag, R. Schekman, A. Kornberg, Proc. Natl.Acad. Sci. U.S.A. 68, 2826 (1971).2. A. Kornberg and T. A. Baker, DNA Replication (Free-man, New York, ed. 2, 1992).3. E. L. Zechner, C. A. Wu, K. J. Marians, J. Biol. Chem.267, 4054 (1992).4. K. Tougu and K. J. Marians, J. Biol. Chem. 271, 21398(1996).5. Y. B. Lu, P. V. Ratnakar, B. K. Mohanty, D. Bastia, Proc.Natl. Acad. Sci. U.S.A. 93, 12902 (1996).6. A. Yuzhakov, Z. Kelman, M. O’Donnell, Cell 96, 153(1999).7. T. Kitani, K. Yoda, T. Ogawa, T. Okazaki, J. Mol. Biol.184, 45 (1985).8. K. J. Marians, Annu. Rev. Biochem. 61, 673 (1992).9. K. Tougu, H. Peng, K. J. Marians, J. Biol. Chem. 269,4675 (1994).10. W. Sun, J. Tormo, T. A. Steitz, G. N. Godson, Proc.Natl. Acad. Sci. U.S.A. 91, 11462 (1994).11. J. L. Keck, D. D. Roche, A. S. Lynch, J. M. Berger, datanot shown.12. T5-overexpression plasmids encoding residues 111 to433 of E. coli DnaG (DnaG-RNAP) preceded by a hexa-histidine tag were constructed and overexpressed inSG13009/pREP4 cells. Cells were lysed by sonicationand the extract was clarified by centrifugation. SolubleDnaG-RNAP was purified by applying the lysate to anickel-affinity column and eluting the protein with 200mM imidazole. His-tagged DnaG-RNAP was further pu-rified by size-exclusion chromatography and concen-trated to ⬎10 mg ml⫺1. Selenomethionine-incorporat-ed protein was expressed as described [G. D. Van Duyne,R. F. Standaert, A. P. Karplus, S. L. Schreiber, J. Clardy, J.Mol. Biol. 229, 105 (1993)] and was purified as per theunsubstituted protein, except that 2 mM dithiothreitolwas included in all purification buffers. ConcentratedHis-tagged DnaG-RNAP was dialyzed against 10 mMHepes (pH 7.5), 100 mM NaCl, and diluted to a finalconcentration of ⬃10 mg ml⫺1before crystallization.Crystals of His-tagged DnaG-RNAP were formed byhanging drop vapor diffusion by mixing 1 ␮l of proteinwith 1 ␮l of well solution [18 to 21% polyethyleneglycol (PEG) 4000, 5% PEG200, 30% ethylene glycol,0.2 M ammonium acetate, 0.05 M sodium acetate (pH5.0), 0.1% dioxane, 2 to 8 mM SrCl2, YCl2, or DyCl3] andequilibrating the drop against 1 ml of well solution atroom temperature for several days. Two nonisomor-phous crystal forms were observed, depending on thedivalent metal that was used; with YCl2or DyCl3, smallplatelike crystals (⬃200 ␮mby100␮mby10␮m) ofsymmetry P212121with unit cell lengths a ⫽ 38, b ⫽56, c ⫽ 140 Å were formed; with SrCl2, thicker bar-shaped crystals (⬃200 ␮mby75␮mby75␮m) ofsymmetry P212121with unit cell lengths a ⫽ 39, b ⫽58, c ⫽ 149 Å were formed.13. Selenomethionine-incorporated SrCl2-based crystalswere solved by MAD phasing to 2.5 Å. Data wereindexed and scaled with MOSFLM [A. G. W. Leslie,Newsletter on Protein Crystallography No. 26 (Scienceand Engineering Research Council, Daresbury Laborato-ry, Warrington, UK, 1992)] and SCALA [W. Kabash,J. Appl. Crystallogr. 21, 916 (1988)]. Selenium siteswere determined with SOLVE [T. C. Terwilliger and J.Berendzen, Acta Crystallogr. D 52, 749 (1996)] andrefined with MLPHARE [Z. Otwinowski, Proc. CCP4Study Weekend (Science and Engineering ResearchCouncil, Daresbury Laboratory, Warrington, UK, 1991),p. 80]. Solvent-flattening with DM [K. Cowtan, JointCCP4 ESF-EACBM Newlett. Protein Crystallogr. 31,34(1994)] yielded readily interpretable electron-densitymaps for model building (Fig. 1B) with O [T. A. Jones,J. Y. Zou, S. W. Cowan, M. Kjeldgaard, Acta Crystallogr.A 47, 110 (1991)]. Elves, an automated structure solu-tion package, was used throughout data analysis andmap construction (J. Holton and T. Alber, in prepara-tion). The initial model was refined against a high-resolution native data set to 1.6 Å resolution with anRworkof 23.1% and an Rfreeof 27.6% by using Refmac/ARP [G. N. Murshudov, A. A. Vagin, E. J. Dodson, ActaCrystallogr. D 53, 240 (1997); V. S. Lamzin and K. S.Wilson, Acta Crystallogr. D 49, 129 (1993)]. The finalmodel includes residues 115 to 428, with the exceptionof residues 192 to 194 and 287, for which electrondensity was not observed. No bond angles for thismodel fall into either disallowed or generously allowedregions of Ramachandrian space.14. The structure of the YCl2-based crystal form wassolved by molecular replacement with AMORE [ J.Navaza, Acta Crystallogr. D 50, 1507 (1994)] and therefined SrCl2structure as an initial model. The mo-lecular replacement solution was refined to 1.7 Åresolution with an Rworkof 20.9% and a Rfreeof26.3% by using Refmac/ARP [G. N. Murshudov, A. A.Vagin, E. J. Dodson, Acta Crystallogr. D 53, 240(1997); V. S. Lamzin and K. S. Wilson, Acta Crystal-logr. D 49, 129 (1993)]. Four Y2⫹ions were modeled,three of which bound in the putative active site ofDnaG-RNAP. The occupancies of these metals wereestimated by using Fo⫺ Fcdifference maps until thedensity for these sites was appropriately accountedfor. The final model included residues from 115 to427, excluding residues 192 to 194, for which elec-tron density was not observed. The rmsd for allcommon C␣atoms between the two structures is 0.6Å. No bond angles for this model fall into eitherdisallowed or generously allowed regions of Ram-achandrian space.15. L. Holm and C. Sander, J. Mol. Biol. 233, 123 (1993).16. L. Aravind, D. D. Leipe, E. V. Koonin, Nucleic Acids Res.26, 4205 (1998).17. J. M. Berger, D. Fass, J. C. Wang, S. C. Harrison, Proc.Natl. Acad. Sci. U.S.A. 95, 7876 (1998).18. J. Versalovic and J. R. Lupski, Gene 136, 281 (1993).19. T. V. Ilyina, A. E. Gorbalenya, E. V. Koonin, J. Mol.Evol. 34, 351 (1992).20. G. Ziegelin, N. A. Linderoth, R. Calendar, E. Lanka, J.Bacteriol. 177, 4333 (1995).21. W. Sun, J. Schoneich, G. N. Godson, J. Bacteriol. 181,3761 (1999).22. M. D. Nichols, K. DeAngelis, J. L. Keck, J. M. Berger,EMBO J. 18, 6177 (1999).23. T. A. Steitz, J. Biol. Chem. 274, 17395 (1999).24. H. H. Egelman, X. Yu, R. Wild, M. M. Hingorani, S. S.Patel, Proc. Natl. Acad. Sci. U.S.A. 92, 3869