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Crystallization of a ribonuclease-resistant fragment of Escherichia coli 5 S ribosomal RNA and its complex with protein L25
J. Mol. Biol. (1983) 171,207-215
Crystallization o f a Ribonuclease-resistant Fragment o f Escherichia coli 5 S Ribosomal R N A and its Complex with Protein L25 SHERIN S. ABDEL-MEGUID 1, PETER B. MOORE1'2 AND THOMAS A. STEITZ 1
1Department of Molecular Biophysics and Biochemistry and 2Department of Chemistry,, Yale University P.O. Box 6666, New Haven, CT 06511, U.S.A. (Received 29 April 1983, and in revised form 23 August 1983) A ribonuclease-resistant fragment of Escherichia coli 5 S ribosomal RNA has been crystallized. The space group is P6122 or P6s22, with a -- 59.5 A and c --- 268 A. The crystals contain one molecule per asymmetric unit, and show diffraction to 4-0 A resolution. Also, a complex of this fragment with L25 ribosomal protein has been crystallized in the same space group, but with a -- 119 A, c -- 250 A and four molecules per asymmetric unit.
1. I n t r o d u c t i o n
5 S RNA is a small ribonucleic acid that constitutes an integral part of the large ribosomal subunit (Rosset et al., 1964). Although it is essential for ribosomal function (Erdmann et al., 1971; Dohme & Nierhaus, 1976), its exact role in protein synthesis remains obscure. In Escherichia coli it is about 39,000 M r and consists of 120 nucleotides (Brownlee et al., 1967). I n vitro, 5 S RNA binds strongly to ribosomal proteins L18 and L25 (Gray et al., 1973) and weakly to L5 (Yu & Wittmann, 1973). In the presence of these three proteins, 5 S RNA also bind specifically to 23 S ribosomal RNA (Gray et al., 1972). Thus, 5 S RNA and its complexes with ribosomal proteins provide a convenient, relatively low molecular weight system for studying the interaction of ribosomal proteins with ribosomal RNAs. For that reason, it has been the object of numerous physical and chemical investigations. (For reviews, see Monier, 1974; Zimmermann, 1980; Garrett et al., 1981.) In the course of their studies of nuclease digestion of 5 S RNAs, Douthwaite et al. (1979) observed that limited exposure to ribonuclease A leads to the formation of a stable fragment consisting of nucleotides 1 to 11, 69 to 87 and 89 to 120. The resistance of this fragment to further nuclease digestion suggests it has a stable structure. Recent nuclear magnetic resonance results indicate t h a t its structure is closely similar to that of the same sequences in the intact 5 S molecule (Kime & Moore, 1983a,b). Moreover, it retains the capability of binding protein 2o7 0022-2836]83]340207-09 $03.00]0 © 1983 AcademicPress Inc. (London)Ltd.
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L25, with which it interacts in a m a n n e r indistinguishable from intact 5 S R N A b y nuclear magnetic resonance criteria (Kime & Moore, 1983c). As a step towards elucidating the three-dimensional structure of the E. coli 5 S R N A and its complexes with ribosomal proteins, we h a v e a t t e m p t e d to crystallize 5 S R N A , its ribonuclease-resistant f r a g m e n t and the complex of f r a g m e n t with ribosomal protein L25. We report here our success in obtaining large, well-shaped crystals of the 5 S R N A f r a g m e n t and its complex with ribosomal protein L25.
2. Materials a n d M e t h o d s
(a) Protein and RNA The 5 S RNA used for these experiments was obtained from E. coil HB101/pKK5-1 (Brosius et al., 1981), which carries the rrnB 5 S RNA cistron on the plasmid pBR322. The 5S RNA used was the product liberated into the eytosol in the presence of chloramphenicol. The details of its preparation have been given elsewhere (Kime & Moore, 1983b). Fragment was prepared from intact 5 S RNA by digesting it with ribonuclease A. The RNA concentration was 1 mg/ml in 0.1 M-KC1, 5 mM-MgCl2, 50 mM-Tris-borate (pH 7"8) and the RNase concentration was 10/Jg/ml. The digestion proceeded for 45 min at 0°C and was terminated by extraction with phenol. The product was purified by chromatography on Sephadex G75 in 0.1 M-NaC1, 3 mM-MgC12, 10 mM-sodium cacodylate (pH 6.0) at 32°C (Kime & Moore, 1983b). The purification of protein L25 has been described (Kime et al., 1981; Kime & Moore, 1982). (b) Electrophoresis Native 5 S RNA, fragment and fragment complex were analyzed by electrophoresis on polyacrylamide gels consisting of 10% (w/v) acrylamide, 0"5% (w/v) bisacrylamide, 0-080/0 (v/v) N,N'-tetraethylmethylenediamine, 0-05% (w/v) ammonium persulfate in 0.1 M-KCI, 5 mM-MgCl2, 50 mM-Tris-borate (pH 7.8). Gels were run at 3 V/cm for 16 h at room temperature and stained with methylene blue, which allows visualization of RNA only (Kime & Moore, 1982). Gel electrophoresis under denaturing conditions was used to examine the internal cleavage patterns in the preparations of fragment. These gels consisted of 11% acrylamide, 0"55% bisacrylamide, 0.08% N,N'-tetraethylmethylenediamine, 0"05% ammonium persulfate in 8 M-urea, 0.9 M-Tris-borate (pH 8-4), 2.5 mM-EDTA. Samples were precipitated with ethanol, dissolved in urea/Tris-borate buffer and heated at 90°C for 2 min before loading on the gels, which were run at 3 V/cm at room temperature overnight.
(c) L25-fragment complex Solutions of fragment and L25 were separately dialyzed into 0.1 M-KC1, 4 mM-MgC12, 5 mM-cacodylic acid (pH 7.2). Their concentrations were estimated from optical density measurements, assuming the extinction of 1 mM-fragment at 260 nm is 424.0 and that a 1 mg/ml solution of L25 has an optical density at 276 nm of 0-38. (The molecular weight of L25 is 10,694; Dovgas et al., 1975; Bitar & Wittmann-Liebold, 1975.) Equimolar mixtures of L25 and fragment were used to form the complex at concentrations around 0"l mM. The resulting solutions were concentrated to about 10 mg/ml by ultrafiltration using Amicon YM-5 membranes.
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(d) Crystallization Before crystallization, concentrated samples were dialyzed against 10mM-sodium cacodylate buffer (pH 7.0), containing 100 mM-NaCl and 3 mM-MgC12 in the case of the RNA fragment, and containing 100 mM-KCIand 4 mM-MgCl2 in the case of the complex. Crystallization conditions were screened utilizing the hanging drop method of vapor diffusion (MePherson, 1976); 5gl of either a 5 mg RNA fragment/ml or a 7.5 mg complex/ml solution were mixed with 5 gl of appropriate components, as detailed in Results, and vapor equilibrated against aqueous solutions of various precipitants such as ethanol, 2-methylpentane-2,4-diol, dioxane, hexane-1, 6-diol, polyethylene glycol 400 (PEG 400), PEG 6000, PEG 20,000 or ammonium sulfate. The crystallization conditions were screened at 4°C or room temperature (18 to 23°C). They were sealed in Styrofoam boxes to reduce the effects of rapid room temperature changes. 3. Results
(a) Crystals of 5 S RNA fragment Large, well-shaped hexagonal bipyramid crystals, as shown in Figure l(a), were obtained under the range of conditions described in Table 1. They grew as isolated crystals achieving dimensions of about 0.4 mm x 0.3 mm × 0.3 mm within three weeks, at room temperature. When they first appear in the crystallization mixtures, they are thin hexagonal plates. They then elongate slowly, normal to the plate plane, to achieve their final bipyramidal shape. Gel electrophoresis of crystals established that the material in the crystals is representative of the RNApresent in the starting sample. A number of crystals of fragment were collected, washed and dissolved for electrophoretic analysis. On non-denaturing gels (Materials and Methods} the crystals proved to contain RNA with electrophoretic mobility identical to that of the original fragment material (data not shown}. RNA from the crystals was also examined on a denaturing gel (Fig. 2(a)), which shows the crystals contain the same RNA fragments as the starting material. The diffraction patterns from these crystals extend to 4.0 A resolution on "stills" with two hours exposure; however, precession photographs show a rapid fall-off in intensity beyond 8 A. A 6 ° precession photograph of the hO1 zone is shown in Figure 3. The space group is P6122 or P6522 with a = 59.5 A and c = 268A. The VM value (Matthews, 1968) is 3.4A3/dalton, assuming one molecule per asymmetric unit. Attempts to grow these crystals at 4°C either failed, produced less regular crystals or produced spherulites (Spencer et al., 1962). The use of ethanol and hexane-l,6-diol produced irregular or microcrystals of the same form. (b) Crystals of 5 S RNA fragment complexed with L25 protein Crystallization of the fragment-L25 complex from methane pentane diol gave two different crystal forms (Fig. l(b) and (c)}, dependent on the magnesium concentration of the crystallization solution (Table 1}. Below 15 mM of added magnesium chloride, crystals grow as hexagonal rods to dimensions of about 0.6 mm × 0.3 m m x 0.3 mm but unfortunately diffract to only 10 A resolution.
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FIo. 1. Single crystals of E. coli ribosomal components. (a) 5 S RNA fragment crystal grown in the presence of 0-25 mM-spermine, 20 mM-MgC12and PEG 400, and equilibrated against 5% PEG 400. (b) and (c) Crystals of 5 S RNA fragment complexed with the L25 protein, grown in the presence of methylpentanediol and (b)10ram or (c) 25 mM-MgC12, and equilibrated against 22% methylpentanediol. Their space group, P6122 or P6s22, is the same as t h a t of crystals of the 5 S R N A fragment. In addition, the lattice constants of these crystals and those of the 5 S RNA fragment crystals are related. The c axis is 250 A, only 7~/o less t h a n t h a t of the fragment, and the a axis is 119 A, exactly double of t h a t of the fragment. Assuming four complex molecules per asymmetric unit gives a V~ value of 2.1 A3/dalton. A 2.5 ° screenless precession photograph is shown in Figure 3(b). The material in these crystals was examined under non-denaturing conditions on a 10~/o polyacrylamide gel (Fig. 2(b)), to establish t h a t t h e y contain R N A protein complex. In the presence of L25, the mobility of fragment is substantially
211
5 S RNA FRAGMENT CRYSTALS TABLE 1
Crystallization conditions that produced large, well-shaped crystals of 5 S R N A fragment and its complex with the L25 protein Components added to the RNA or complex
RNA or complex equilibrated against
A. 5 S RNA fragment 0.2 M-NaCI, 4 to 6% PEG 400 0 to 1.0 mM-sperminehydrochloride, 4 to 6% PEG 400 (2) 10mM-eacodylate(pH 7"0), 0.2 M-NaCI, 1% PEG 6000 or 20,000 15 mM-MgCI2, 1% PEG 6000 or 20,000 (3) 10 mM-caeodylate (pH 7.0) 0-2 M-NaCi, 15 mM-MgCI~, 10% dioxane 10% dioxane (4) 10 mM-eacodylate (pH 7.0) 0.2 M-NaCI, 15 mM-MgCi2, 6% MPD 6% MPDt B. 5 S RNA fragment complexedwith L25 protein (1) 10 mM-cacodylate (pH 7.0), 0.2 M-KCI, 5 to 10 mM-MgCI2, 18 to 24% MPD 18 to 24% MPD (2) 10 mM-eacodylate (pH 7.0), 0-2 M-KCI, 5 to 15 mM-MgCI2, 160 PEG 400 16% PEG 400 (3) 10 mM-cacodylate (pH 7.0), 0.2 M-KCI, 15 to 30 mM-MgCI2, 16 to 24% MPD 16 to 24% MPD (1) 10 mM-caeodylate (pH 7-0), 15 to 30 mM-MgCI2,
Morphology Hexagonal bipyramids Hexagonal bipyramids Hexagonal bipyramids Hexagonal bipyramids Hexagonal rods Hexagonal rods Tetragonal bipyramids
t MPD, methylpentanediol.
reduced (see e.g. K i m e & Moore, 1983e). Lanes 1 a n d 2 of Figure 2(b) show this effect for n o r m a l f r a g m e n t and f r a g m e n t plus L25. The material in lane 3 was t a k e n from crystals of complex; the mobility of the R N A in the crystals is the same as t h a t of f r a g m e n t - L 2 5 complex. F r a g m e n t - L 2 5 complex f o r m s t e t r a g o n a l b i p y r a m i d crystals ( 0 . 2 m m × 0.15 m m × 0-15 ram) a b o v e 15 mM of a d d e d m a g n e s i u m chloride. These crystals are v e r y unstable to X - r a y irradiation and to changes of t e m p e r a t u r e a n d m o t h e r liquor and thus h a v e not been analyzed crystallographically. B o t h the hexagonal rod and t e t r a g o n a l b i p y r a m i d crystals are grown a t r o o m t e m p e r a t u r e . The t e t r a g o n a l b i p y r a m i d crystals can also be grown a t 4°C.
4. D i s c u s s i o n
I n the course of this work, crystallization of i n t a c t 5 S R N A was a t t e m p t e d w i t h o u t success. Only Morikawa et al. (1982) h a v e so far reported success in growing crystals of intact 5 S R N A from a thermophilic bacterium. Their crystals
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1
2
3
(a)
4
P. B. M O O R E A N D T. A. S T E I T Z
1
2
3
(b)
FIo. 2. Gel electrophoresis analysis of crystals. (a) A denaturing gel (see Materials and Methods), run to examine the state of the 5 S RNA fragment material following crystallization. Lane 1 shows undigested 5 S RNA. Lane 2 is a standard fragment preparation showing (from top to bottom) bands corresponding to the following oligonucleotides: bases (69-120), bases (89-120), bases (69-87) and bases (1-11) (Douthwaite et al., 1979). Lane 3 is the sample used for crystallization before crystallization, and lane 4 is material taken from crystals. (The slight smearing of bands in lane 4 represents a technical problem with the gel in question.) (b) A native gel (see Materials and Methods) was run to analyze the material crystallized from mixtures containing fragment-L25 complexes. Lane 1 : 5 S fragment. Lane 2, 5 S fragment with L25 added. Lane 3: material derived from washed crystals.
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CRYSTALS
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FIG. 3. X-ray diffraction photographs. (a) A 6 ° precession photograph of the hOl zone of a 5 S RNA fragment crystal exposed for l0 h. (b) A 2.5 ° screenless precession photograph of a hexagonal rod crystal of 5 S RNA fragment-L25 complex, exposed for 2 h. Both photographs were obtained using a GX6 Elliott rotating anode generator, run at 50 mA, 35 kV, with a 200/zm focal cup.
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diffract, however, to only 25 A resolution and offer no hope for the elucidation of the structure. Thus, it is clear t h a t a structure determination of the 5 S fragment, even at intermediate resolution (4 to 5 A), could contribute significantly to the ultimate goal of the detailed structure determination of the 5 S R N A molecule. The presence of two helical segments in the secondary structure of the fragment (Douthwaite et al., 1979; Kime & Moore, 1983b) and the knowledge of its sequence (Brownlee et al., 1967) should facilitate the task of model building this molecule into a well-phased electron density map at 4 to 5 A resolution. Our general experience in crystallizing the 5 S R N A fragment and its protein complex indicate t h a t further refinement of our current crystallization conditions and techniques for handling of the crystals could improve the resolution with which t h e y diffract. I t is hoped t h a t this report will serve as a stimulus for further studies on improving the resolution of these crystals or obtaining other, better diffracting crystals. We are grateful for encouragement and advice from Dr Harold W. Wyckoff and for technical assistance from Mrs Betty Rennie and Mrs Grace Sun. This work was supported by a National Institutes of Health grant (AI-09167 to P.B.M.) and by a U.S. Public Health Services grant (GM-22778).
REFERENCES Bitar, K. G. & Wittmann-Liebold, B. (1975). Hoppe-Seyler's Z. Physiol. Chem. 356, 13431352. Brosuis, J., Dull, T. J., Sleeter, D. D. & Noller, H. F. (1981}. J. Mol. Biol. 148, 107-127. Brownlee, G. G., Sanger, F. & Barrell, B. G. (1967). Nature (London), 215, 735-736. Dohme, F. & Nierhaus, K. H. (1976). Proc. Nat. Acad. Sci., U.S.A. 73, 2221-2225. Douthwaite, S., Garrett, R. A., .Wagner, R. & Feunteun, J. (1979}. Nucl. Acids Res. 6, 2453-2470. Dovgas, N. V., Markova, L. F., Mednikova, T. A., Vinokurov, L. M., Alakhov, Yu. B. & Ovchinnikov, Yu. A. (1975). F E B S Letters, 53, 351-354. Erdmann, V. A., Fahnestock, S., Higo, K. & Nomura, M. (1971}. Proc. Nat. Acad. Sci., U.S.A. 68, 2932-2936. Garrett, R. A., Douthwaite, S. & Noller, H. F. (1981). Trends Biochem. Sci. 6, 137-139. Gray, P. N., Bellemare, G. & Monier, R. (1972). F E B S Letters, 24, 156-160. Gray, P. N., Bellemare, G., Monier, R., Garrett, R. A. & StSffler, G. (1973). J. Mol. Biol. 77, 133-152. Kime, M. J. & Moore, P. B. (1982). Nucl. Acids Res. 10, 4973-4983. Kime, M. J. & Moore, P. B. (1983a). F E B S Letters, 153, 199-203. Kime, M. J. & Moore, P. B. (1983b). Biochemistry, 22, 2615-2621. Kime, M. J. & Moore, P. B. (1983c). Biochemistry, 22, 2622-2629. Kime, M. J., Ratcliffe, R. G., Moore, P. B. & Williams, R. J. P. (1981). Eur. J. Biochem. 116, 269-276. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. McPherson, A. Jr (1976). Methods Biochem. Anal. 23, 249-345. Monier, R. (1974). In Ribosomes (Nomura, M., Tissieres, A. & Lengyel, P., eds), pp. 141168, Cold Spring Harbor Laboratory, Cold Spring Harbor. Morikawa, K., Makoto, K. & Takamura, S. (1982). F E B S Letters, 145, 194-196. Rosset, R., Monier, R. & Julien, J. (1964). Bull. Soc. Chim. Biol. 46, 87-109. Spencer, M., Fuller, W., Wilkins, M. H. F. & Brown, G. L. (1962). Nature (London), 194, 1014-1020.
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Yu, R. S. T. & Wittmann, H. G. (1973). Biochim. Biophys. Acta, 324, 375-385. Zimmermann, R. A. (1980). In Ribosomes Structure, Function & Genetics (Chambliss, G., Craven, G. R., Davies, J., Davis, K., Kahan, L. & Nomura, M., eds), pp. 135-170, University Park Press, Baltimore.
Edited by R. Huber
Crystallization o f a Ribonuclease-resistant Fragment o f Escherichia coli 5 S Ribosomal R N A and its Complex with Protein L25 SHERIN S. ABDEL-MEGUID 1, PETER B. MOORE1'2 AND THOMAS A. STEITZ 1
1Department of Molecular Biophysics and Biochemistry and 2Department of Chemistry,, Yale University P.O. Box 6666, New Haven, CT 06511, U.S.A. (Received 29 April 1983, and in revised form 23 August 1983) A ribonuclease-resistant fragment of Escherichia coli 5 S ribosomal RNA has been crystallized. The space group is P6122 or P6s22, with a -- 59.5 A and c --- 268 A. The crystals contain one molecule per asymmetric unit, and show diffraction to 4-0 A resolution. Also, a complex of this fragment with L25 ribosomal protein has been crystallized in the same space group, but with a -- 119 A, c -- 250 A and four molecules per asymmetric unit.
1. I n t r o d u c t i o n
5 S RNA is a small ribonucleic acid that constitutes an integral part of the large ribosomal subunit (Rosset et al., 1964). Although it is essential for ribosomal function (Erdmann et al., 1971; Dohme & Nierhaus, 1976), its exact role in protein synthesis remains obscure. In Escherichia coli it is about 39,000 M r and consists of 120 nucleotides (Brownlee et al., 1967). I n vitro, 5 S RNA binds strongly to ribosomal proteins L18 and L25 (Gray et al., 1973) and weakly to L5 (Yu & Wittmann, 1973). In the presence of these three proteins, 5 S RNA also bind specifically to 23 S ribosomal RNA (Gray et al., 1972). Thus, 5 S RNA and its complexes with ribosomal proteins provide a convenient, relatively low molecular weight system for studying the interaction of ribosomal proteins with ribosomal RNAs. For that reason, it has been the object of numerous physical and chemical investigations. (For reviews, see Monier, 1974; Zimmermann, 1980; Garrett et al., 1981.) In the course of their studies of nuclease digestion of 5 S RNAs, Douthwaite et al. (1979) observed that limited exposure to ribonuclease A leads to the formation of a stable fragment consisting of nucleotides 1 to 11, 69 to 87 and 89 to 120. The resistance of this fragment to further nuclease digestion suggests it has a stable structure. Recent nuclear magnetic resonance results indicate t h a t its structure is closely similar to that of the same sequences in the intact 5 S molecule (Kime & Moore, 1983a,b). Moreover, it retains the capability of binding protein 2o7 0022-2836]83]340207-09 $03.00]0 © 1983 AcademicPress Inc. (London)Ltd.
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L25, with which it interacts in a m a n n e r indistinguishable from intact 5 S R N A b y nuclear magnetic resonance criteria (Kime & Moore, 1983c). As a step towards elucidating the three-dimensional structure of the E. coli 5 S R N A and its complexes with ribosomal proteins, we h a v e a t t e m p t e d to crystallize 5 S R N A , its ribonuclease-resistant f r a g m e n t and the complex of f r a g m e n t with ribosomal protein L25. We report here our success in obtaining large, well-shaped crystals of the 5 S R N A f r a g m e n t and its complex with ribosomal protein L25.
2. Materials a n d M e t h o d s
(a) Protein and RNA The 5 S RNA used for these experiments was obtained from E. coil HB101/pKK5-1 (Brosius et al., 1981), which carries the rrnB 5 S RNA cistron on the plasmid pBR322. The 5S RNA used was the product liberated into the eytosol in the presence of chloramphenicol. The details of its preparation have been given elsewhere (Kime & Moore, 1983b). Fragment was prepared from intact 5 S RNA by digesting it with ribonuclease A. The RNA concentration was 1 mg/ml in 0.1 M-KC1, 5 mM-MgCl2, 50 mM-Tris-borate (pH 7"8) and the RNase concentration was 10/Jg/ml. The digestion proceeded for 45 min at 0°C and was terminated by extraction with phenol. The product was purified by chromatography on Sephadex G75 in 0.1 M-NaC1, 3 mM-MgC12, 10 mM-sodium cacodylate (pH 6.0) at 32°C (Kime & Moore, 1983b). The purification of protein L25 has been described (Kime et al., 1981; Kime & Moore, 1982). (b) Electrophoresis Native 5 S RNA, fragment and fragment complex were analyzed by electrophoresis on polyacrylamide gels consisting of 10% (w/v) acrylamide, 0"5% (w/v) bisacrylamide, 0-080/0 (v/v) N,N'-tetraethylmethylenediamine, 0-05% (w/v) ammonium persulfate in 0.1 M-KCI, 5 mM-MgCl2, 50 mM-Tris-borate (pH 7.8). Gels were run at 3 V/cm for 16 h at room temperature and stained with methylene blue, which allows visualization of RNA only (Kime & Moore, 1982). Gel electrophoresis under denaturing conditions was used to examine the internal cleavage patterns in the preparations of fragment. These gels consisted of 11% acrylamide, 0"55% bisacrylamide, 0.08% N,N'-tetraethylmethylenediamine, 0"05% ammonium persulfate in 8 M-urea, 0.9 M-Tris-borate (pH 8-4), 2.5 mM-EDTA. Samples were precipitated with ethanol, dissolved in urea/Tris-borate buffer and heated at 90°C for 2 min before loading on the gels, which were run at 3 V/cm at room temperature overnight.
(c) L25-fragment complex Solutions of fragment and L25 were separately dialyzed into 0.1 M-KC1, 4 mM-MgC12, 5 mM-cacodylic acid (pH 7.2). Their concentrations were estimated from optical density measurements, assuming the extinction of 1 mM-fragment at 260 nm is 424.0 and that a 1 mg/ml solution of L25 has an optical density at 276 nm of 0-38. (The molecular weight of L25 is 10,694; Dovgas et al., 1975; Bitar & Wittmann-Liebold, 1975.) Equimolar mixtures of L25 and fragment were used to form the complex at concentrations around 0"l mM. The resulting solutions were concentrated to about 10 mg/ml by ultrafiltration using Amicon YM-5 membranes.
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(d) Crystallization Before crystallization, concentrated samples were dialyzed against 10mM-sodium cacodylate buffer (pH 7.0), containing 100 mM-NaCl and 3 mM-MgC12 in the case of the RNA fragment, and containing 100 mM-KCIand 4 mM-MgCl2 in the case of the complex. Crystallization conditions were screened utilizing the hanging drop method of vapor diffusion (MePherson, 1976); 5gl of either a 5 mg RNA fragment/ml or a 7.5 mg complex/ml solution were mixed with 5 gl of appropriate components, as detailed in Results, and vapor equilibrated against aqueous solutions of various precipitants such as ethanol, 2-methylpentane-2,4-diol, dioxane, hexane-1, 6-diol, polyethylene glycol 400 (PEG 400), PEG 6000, PEG 20,000 or ammonium sulfate. The crystallization conditions were screened at 4°C or room temperature (18 to 23°C). They were sealed in Styrofoam boxes to reduce the effects of rapid room temperature changes. 3. Results
(a) Crystals of 5 S RNA fragment Large, well-shaped hexagonal bipyramid crystals, as shown in Figure l(a), were obtained under the range of conditions described in Table 1. They grew as isolated crystals achieving dimensions of about 0.4 mm x 0.3 mm × 0.3 mm within three weeks, at room temperature. When they first appear in the crystallization mixtures, they are thin hexagonal plates. They then elongate slowly, normal to the plate plane, to achieve their final bipyramidal shape. Gel electrophoresis of crystals established that the material in the crystals is representative of the RNApresent in the starting sample. A number of crystals of fragment were collected, washed and dissolved for electrophoretic analysis. On non-denaturing gels (Materials and Methods} the crystals proved to contain RNA with electrophoretic mobility identical to that of the original fragment material (data not shown}. RNA from the crystals was also examined on a denaturing gel (Fig. 2(a)), which shows the crystals contain the same RNA fragments as the starting material. The diffraction patterns from these crystals extend to 4.0 A resolution on "stills" with two hours exposure; however, precession photographs show a rapid fall-off in intensity beyond 8 A. A 6 ° precession photograph of the hO1 zone is shown in Figure 3. The space group is P6122 or P6522 with a = 59.5 A and c = 268A. The VM value (Matthews, 1968) is 3.4A3/dalton, assuming one molecule per asymmetric unit. Attempts to grow these crystals at 4°C either failed, produced less regular crystals or produced spherulites (Spencer et al., 1962). The use of ethanol and hexane-l,6-diol produced irregular or microcrystals of the same form. (b) Crystals of 5 S RNA fragment complexed with L25 protein Crystallization of the fragment-L25 complex from methane pentane diol gave two different crystal forms (Fig. l(b) and (c)}, dependent on the magnesium concentration of the crystallization solution (Table 1}. Below 15 mM of added magnesium chloride, crystals grow as hexagonal rods to dimensions of about 0.6 mm × 0.3 m m x 0.3 mm but unfortunately diffract to only 10 A resolution.
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FIo. 1. Single crystals of E. coli ribosomal components. (a) 5 S RNA fragment crystal grown in the presence of 0-25 mM-spermine, 20 mM-MgC12and PEG 400, and equilibrated against 5% PEG 400. (b) and (c) Crystals of 5 S RNA fragment complexed with the L25 protein, grown in the presence of methylpentanediol and (b)10ram or (c) 25 mM-MgC12, and equilibrated against 22% methylpentanediol. Their space group, P6122 or P6s22, is the same as t h a t of crystals of the 5 S R N A fragment. In addition, the lattice constants of these crystals and those of the 5 S RNA fragment crystals are related. The c axis is 250 A, only 7~/o less t h a n t h a t of the fragment, and the a axis is 119 A, exactly double of t h a t of the fragment. Assuming four complex molecules per asymmetric unit gives a V~ value of 2.1 A3/dalton. A 2.5 ° screenless precession photograph is shown in Figure 3(b). The material in these crystals was examined under non-denaturing conditions on a 10~/o polyacrylamide gel (Fig. 2(b)), to establish t h a t t h e y contain R N A protein complex. In the presence of L25, the mobility of fragment is substantially
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5 S RNA FRAGMENT CRYSTALS TABLE 1
Crystallization conditions that produced large, well-shaped crystals of 5 S R N A fragment and its complex with the L25 protein Components added to the RNA or complex
RNA or complex equilibrated against
A. 5 S RNA fragment 0.2 M-NaCI, 4 to 6% PEG 400 0 to 1.0 mM-sperminehydrochloride, 4 to 6% PEG 400 (2) 10mM-eacodylate(pH 7"0), 0.2 M-NaCI, 1% PEG 6000 or 20,000 15 mM-MgCI2, 1% PEG 6000 or 20,000 (3) 10 mM-caeodylate (pH 7.0) 0-2 M-NaCi, 15 mM-MgCI~, 10% dioxane 10% dioxane (4) 10 mM-eacodylate (pH 7.0) 0.2 M-NaCI, 15 mM-MgCi2, 6% MPD 6% MPDt B. 5 S RNA fragment complexedwith L25 protein (1) 10 mM-cacodylate (pH 7.0), 0.2 M-KCI, 5 to 10 mM-MgCI2, 18 to 24% MPD 18 to 24% MPD (2) 10 mM-eacodylate (pH 7.0), 0-2 M-KCI, 5 to 15 mM-MgCI2, 160 PEG 400 16% PEG 400 (3) 10 mM-cacodylate (pH 7.0), 0.2 M-KCI, 15 to 30 mM-MgCI2, 16 to 24% MPD 16 to 24% MPD (1) 10 mM-caeodylate (pH 7-0), 15 to 30 mM-MgCI2,
Morphology Hexagonal bipyramids Hexagonal bipyramids Hexagonal bipyramids Hexagonal bipyramids Hexagonal rods Hexagonal rods Tetragonal bipyramids
t MPD, methylpentanediol.
reduced (see e.g. K i m e & Moore, 1983e). Lanes 1 a n d 2 of Figure 2(b) show this effect for n o r m a l f r a g m e n t and f r a g m e n t plus L25. The material in lane 3 was t a k e n from crystals of complex; the mobility of the R N A in the crystals is the same as t h a t of f r a g m e n t - L 2 5 complex. F r a g m e n t - L 2 5 complex f o r m s t e t r a g o n a l b i p y r a m i d crystals ( 0 . 2 m m × 0.15 m m × 0-15 ram) a b o v e 15 mM of a d d e d m a g n e s i u m chloride. These crystals are v e r y unstable to X - r a y irradiation and to changes of t e m p e r a t u r e a n d m o t h e r liquor and thus h a v e not been analyzed crystallographically. B o t h the hexagonal rod and t e t r a g o n a l b i p y r a m i d crystals are grown a t r o o m t e m p e r a t u r e . The t e t r a g o n a l b i p y r a m i d crystals can also be grown a t 4°C.
4. D i s c u s s i o n
I n the course of this work, crystallization of i n t a c t 5 S R N A was a t t e m p t e d w i t h o u t success. Only Morikawa et al. (1982) h a v e so far reported success in growing crystals of intact 5 S R N A from a thermophilic bacterium. Their crystals
212
S. S. A B D E L - M E G U I D ,
1
2
3
(a)
4
P. B. M O O R E A N D T. A. S T E I T Z
1
2
3
(b)
FIo. 2. Gel electrophoresis analysis of crystals. (a) A denaturing gel (see Materials and Methods), run to examine the state of the 5 S RNA fragment material following crystallization. Lane 1 shows undigested 5 S RNA. Lane 2 is a standard fragment preparation showing (from top to bottom) bands corresponding to the following oligonucleotides: bases (69-120), bases (89-120), bases (69-87) and bases (1-11) (Douthwaite et al., 1979). Lane 3 is the sample used for crystallization before crystallization, and lane 4 is material taken from crystals. (The slight smearing of bands in lane 4 represents a technical problem with the gel in question.) (b) A native gel (see Materials and Methods) was run to analyze the material crystallized from mixtures containing fragment-L25 complexes. Lane 1 : 5 S fragment. Lane 2, 5 S fragment with L25 added. Lane 3: material derived from washed crystals.
5 S RNA FRAGMENT
CRYSTALS
213
FIG. 3. X-ray diffraction photographs. (a) A 6 ° precession photograph of the hOl zone of a 5 S RNA fragment crystal exposed for l0 h. (b) A 2.5 ° screenless precession photograph of a hexagonal rod crystal of 5 S RNA fragment-L25 complex, exposed for 2 h. Both photographs were obtained using a GX6 Elliott rotating anode generator, run at 50 mA, 35 kV, with a 200/zm focal cup.
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S. S. ABDEL-MEGUID, P. B. MOORE AND T. A. STEITZ
diffract, however, to only 25 A resolution and offer no hope for the elucidation of the structure. Thus, it is clear t h a t a structure determination of the 5 S fragment, even at intermediate resolution (4 to 5 A), could contribute significantly to the ultimate goal of the detailed structure determination of the 5 S R N A molecule. The presence of two helical segments in the secondary structure of the fragment (Douthwaite et al., 1979; Kime & Moore, 1983b) and the knowledge of its sequence (Brownlee et al., 1967) should facilitate the task of model building this molecule into a well-phased electron density map at 4 to 5 A resolution. Our general experience in crystallizing the 5 S R N A fragment and its protein complex indicate t h a t further refinement of our current crystallization conditions and techniques for handling of the crystals could improve the resolution with which t h e y diffract. I t is hoped t h a t this report will serve as a stimulus for further studies on improving the resolution of these crystals or obtaining other, better diffracting crystals. We are grateful for encouragement and advice from Dr Harold W. Wyckoff and for technical assistance from Mrs Betty Rennie and Mrs Grace Sun. This work was supported by a National Institutes of Health grant (AI-09167 to P.B.M.) and by a U.S. Public Health Services grant (GM-22778).
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Yu, R. S. T. & Wittmann, H. G. (1973). Biochim. Biophys. Acta, 324, 375-385. Zimmermann, R. A. (1980). In Ribosomes Structure, Function & Genetics (Chambliss, G., Craven, G. R., Davies, J., Davis, K., Kahan, L. & Nomura, M., eds), pp. 135-170, University Park Press, Baltimore.
Edited by R. Huber