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Crystallization Papers Crystallization and Preliminary Crystallographic crystallization papers Acta Crystallographica Section D Biological Crystallization and preliminary crystallographic Crystallography analysis of T7 RNA polymerase elongation complex ISSN 0907-4449 Dmitry Temiakov,a² Tahir H. Stable transcription-elongation complexes consisting of T7 RNA Received 29 July 2002 Tahirov,b² Michael Anikin,a polymerase (molecular mass 99 kDa) in association with a nucleic Accepted 29 October 2002 William T. McAllister,a acid scaffold consisting of an 8 bp RNA±DNA hybrid and 10 bp of c,d downstream DNA were assembled and crystallized by the sitting- Dmitry G. Vassylyev * and drop vapour-diffusion technique under near-physiological conditions. c,d,e,f Shigeyuki Yokoyama * The crystals diffract beyond 2.6 AÊ resolution and belong to space group P1, with unit-cell parameters a = 79.91, b = 84.97, c = 202 AÊ , = 90.36, = 92.97, = 109.94 . An unambiguous molecular- a Morse Institute of Molecular Genetics, replacement solution was found using the C-terminal portion of the Department of Microbiology and Immunology, T7 RNA polymerase structure from the early initiation complex as a SUNY Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, New York search model. Model building and structure re®nement are now in 11203-2098, USA, bHigh Throughput Factory, progress. RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan, cCellular Signaling Laboratory, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, 1. Introduction Temiakov et al., 2002; Place et al., 2000; Sousa et d Sayo, Hyogo 679-5148, Japan, Structurome al., 1992; Temiakov et al., 2000). Project, RIKEN Harima Institute at SPring-8, Although the DNA-dependent RNA poly- merase (RNAP) encoded by bacteriophage T7 Four structures of T7 RNAP have been 1-1-1 Kouto, Mikazuki-cho, Sayo, reported: free enzyme, RNAP bound to the Hyogo 679-5148, Japan, eDepartment of consists of a single subunit (883 amino acids, transcription inhibitor T7 lysozyme, a binary Biophysics and Biochemistry, Graduate School 99 kDa), this enzyme is able to carry out all the of Science, Univeristy of Tokyo, 7-3-1 Hongo, steps of the transcription cycle in the same RNAP±promoter complex and an early IC in Bukyo-ku, Tokyo 113-0033, Japan, and fRIKEN manner as the multisubunit RNAPs found in which the ®rst three bases of the template Gemomic Sciences Center, 1-7-22 Suehiro-cho, bacteria and eukaryotic cells (McAllister, strand have been transcribed (Cheetham et al., Tsurumi, Yokohama 230-0045, Japan 1997). The C-terminal portion of phage RNAP 1999; Cheetham & Steitz, 1999; Jeruzalmi & (residues 267±883, which contains the active Steitz, 1998; Sousa et al., 1993). All these site) is structurally related to the pol I-like structures exhibit a similar conformation of the ² These authors contributed equally to this enzyme, indicating that the initial stages of work. DNA polymerases and it has been suggested that the N-terminal domain of the phage transcription may be achieved without serious RNAP confers upon the catalytic core the changes in enzyme structure. However, these structures were not in agreement with Correspondence e-mail: ability to carry out the steps that are speci®c to [email protected], the transcription process (Cheetham et al., biochemical observations with regard to the [email protected] 1999; Cheetham & Steitz, 2000; Jeruzalmi & organization of the EC and did not provide Steitz, 1998; Sousa et al., 1993). plausible mechanisms for further transcription To initiate RNA synthesis, the enzyme must progress (Huang & Sousa, 2000; Liu & Martin, bind to a speci®c promoter DNA sequence that 2001; Ma et al., 2002; Severinov, 2000; lies upstream of the start site for transcription, Temiakov et al., 2000). melt apart the two strands of the DNA in the Here, we report crystallization of the T7 vicinity of the start site (forming a transcription RNAP EC, the structure of which may help to bubble) and begin RNA synthesis using the resolve the principal differences between coding strand of the downstream DNA as a previous biochemical and structural data template and a single ribonucleotide as a (Cheetham & Steitz, 1999; Severinov, 2000; primer. A number of biochemical observations Temiakov et al., 2000) and provide further indicate that the organization of the tran- insight into mechanisms of transcription at scription complex during elongation (EC) is various stages of transcription cycle. distinct from the organization of the early initiation complex (IC), suggesting that 2. Experimental procedures and results substantial changes in structure may occur during this transition. These include changes in 2.1. T7 RNAP purification and assembly of the the footprint of the enzyme on the DNA, elongation complex changes in the pattern of protease cleavage Histidine-tagged T7 RNAP was puri®ed as and changes in nucleic acid±protein contacts as previously described (He et al., 1997; Temiakov probed by nuclease protection and protein± et al., 2000) with an additional puri®cation step # 2003 International Union of Crystallography nucleic acid crosslinking experiments (Brieba on phosphocellulose introduced to ensure high Printed in Denmark ± all rights reserved & Sousa, 2001; Ikeda & Richardson, 1986; Ma, purity of the enzyme. Brie¯y, after elution from Acta Cryst. (2003). D59, 185±187 Temiakov et al. T7 RNA polymerase elongation complex 185 crystallization papers Ni2+-agarose beads the protein was dialyzed complexes were assembled by mixing T7 Table 1 against phosphate buffer (20 mM sodium RNAP with the scaffold in a 1:1 molar ratio Data-collection statistics for the T7 RNAP elonga- tion complex. phosphate pH 7.7, 50 mM NaCl, 5 mM in buffer containing 10 mM Tris pH 8.1, -mercaptoethanol) and mixed with 1 ml of 200 mM NaCl, 20 mM MgCl2 and 5 mM Values in parentheses are for the highest resolution shell. phosphocellulose slurry (Whatman P11; -mercaptoethanol. Such complexes have Unit-cell parameters prepared as recommended by the supplier). been shown to be stable and fully active in a (AÊ ) 79.91 After 1 h incubation on a rotary shaker at vitro and to have all of the properties of ECs b (AÊ ) 84.97 Ê 277 K, the slurry was ®ltered through an formed by initiation at a T7 promoter in a c (A) 202.00 () 90.36 empty PD10 column (Pharmacia). The double-stranded DNA template (Temiakov () 92.97 ¯owthrough was discarded and the poly- et al., 2003). () 109.94 Temperature (K) 100 merase was eluted with four 1 ml aliquots of Space group P1 elution buffer (200 mM sodium phosphate Molecules in asymmetric unit 4 pH 7.7, 400 mM NaCl, 5 mM -mercapto- 2.2. Crystallization Solvent content (%) 57.3 Resolution (AÊ ) 40±2.6 (2.69±2.6) ethanol). Peak fractions were identi®ed by We elected to use low-salt solutions in Observations 488245 gel electrophoresis, pooled and dialyzed search of crystallization conditions for the Unique re¯ections 143689 against buffer containing 10 mM Tris pH 8.1, Rmerge² 0.081 (0.451) T7 RNAP EC in order to ensure stability of Completeness (%) 94.2 (73.6) 200 mM NaCl, 20 mM MgCl2 and 5 mM the assembled complex (Temiakov et al., I/(I) 14.7 (2.9) -mercaptoethanol. The protein was 2003). In previous crystallization attempts 1 ² R = I I = I , where I is the intensity of concentrated to 50 mg ml on a Vivaspin (Chen et al., 1999), it was demonstrated that merge j j h jij h ji j re¯ection j and Ij is the average intensity of re¯ection j. concentrator (Sartorius), divided into PEG 8000 was the only precipitant that P h i P aliquots and stored at 203 K. We estimate produced crystals of wild-type and His- the purity of the polymerase to be greater tagged T7 polymerase. The same precipitant ethanol, 100 mM Tris buffer pH 8.1) yielded than 99% by gel electrophoresis. was successfully used in crystallization of high-quality crystals (Fig. 2). Synthetic DNA oligonucleotides (18 nt promoter and initiation complexes of T7 template strand and 10 nt non-template RNAP, even though a higher salt concen- 2.3. Data collection and processing strand) and 12-mer synthetic RNA (Dhar- tration was used (Cheetham & Steitz, 1999; The quality of the crystals was ®rst magon Research) were dissolved in water Jeruzalmi & Steitz, 1998). We have tried a checked using a laboratory X-ray generator (all at 3 mM concentration), incubated for variety of polyethylene glycol precipitants (Rigaku FR-D, rotating anode, Cu radiation, 7 min at 343 K and then allowed to cool to and found that solutions of PEG 5000 MME, focal size 0.15 1.5 mm, confocal mirrors) 293 K over a period of 2±3 h to give a PEG 6000 and PEG 8000 produced crystals, operated at 50Â kV and 100 mA. Diffraction solution of scaffold template having an the latter crystals being suitable for crystal- was measured using a Rigaku R-AXIS IV++ eight-base-pair RNA±DNA hybrid with an lographic studies. Crystallization was carried imaging-plate detector. For data collection unpaired 5 RNA `tail' of 4 nt and 10 bp of 0 out by the sitting-drop vapour-diffusion at room temperature (293 K), the crystals downstream DNA (Fig. 1a). Transcription technique at 293 K. The drop, containing 1 were mounted in glass capillaries. For data 2 mlof10mgml complex solution, was collection at cryotemperature (100 K), the mixed with 2 ml of well solution containing crystals were soaked in a stock solution in 10% PEG 8000, 10% glycerol, 5 mM which the concentration of cryoprotectant -mercaptoethanol, 100 mM Tris buffer pH was gradually increased over a period of 8.1. Thin plate-like crystals (0.35 0.35 Â Â 2±3 min.
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