X-ray crystallographic studies of polymorphic forms of yeast phenylalanine transfer RNA

J. Mol. Biol. (1973) 75, 421-428

X-ray Crystallographic Studies of Polymorphic Forms of Yeast Phenylalanine Transfer RNA S.H.Kmr,G.

&UIGLEY,P. L. SUDDATH, A. MCPHERSON,D. SNEDEN, J. J. KIM, J. WEINZIERL AND ALEXANDER RICH Department of Biology Maseachwette Institute of Technology Cambridge, Mass. 02139, U.S.A. (Received 25 September 1972)

Yeast phenylalanine transfer RNA has been found to crystallize in five different crystal systems involving eight different space groups. The X-ray diffraction characteristics of these forms are described. One of the orthorhombic forms yields a diffraction pattern with higher resolution than either the hexagonal, the cubic or the monoclinic forms. One region of this orthorhombic diffraction pattern is particularly sensitive to X-ray exposure and to changes in the concentration of various solutes. The diffraction p&tern from the cubic crystal form extends to a resolution of 3 A, and there are a number of strong reflections in the 3 to 4 A region which suggest that double-helical segments of the tRNA molecules are oriented along the 4-fold axes. Some comments are made regarding the nature of the polymorphism in the transfer RNA crystals.

1. Introduction Transfer RNA is a molecule which is involved in the translation of the nucleotide sequence information in messenger RNA into the polypeptide sequences in protein molecules. Although some general details of this process are known, any detailed description of the function of tRNA is impossible without a knowledge of its threedimensional structure. This problem is now being attacked by a number of laboratories using X-ray crystallographic techniques (Young et al., 1969; Blake et al., 1970; Cramer et al., 1970; Kim et al., 1971; Mirzabekov et al., 1972; Schevitz et al., 1972). A variety of different crystallographic forms has been discovered for different tRNAs. Our efforts in this field have focused on the phenylalanine tRNA from brewer’s yeast and in an earlier report we have shown that it is possible to obtain crystals of this tRNA which yield 2.3 A resolution X-ray diffraction data in an orthorhombic unit cell (Kim et al., 1971). We have continued to study the crystallographic characteristics of this transfer RNA and we report here on its unusual polymorphism. With only slight changes in the conditions of crystallization the phenylalanine tRNA has been crystallized in five different crystal systems using eight different space groups. In the present paper we describe methods for producing seven of these eight space groups and report on their characteristics. They differ considerably both in terms of the resolution of their X-ray diffraction patterns and in the symmetry elements used in the molecular packing. We also describe some unusual changes of 421

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diffraction intensities which occur in one of these crystal forms when preformed crystals are subjected to slight changes in solvent composition or to X-ray exposure.

2. Materials and Methods (a) Methods of crystdizath The method of crystallization has been briefly described elsewhere (Kim et al., 1971). Purified yeast phenylalanine transfer RNA (Boehringer-Mannheim) was dissolved in distilled water to a concentration of 10 mg/ml and dialyzed extensively against 2 mM-MgCl,. This tRNA solution was then precipitated with 2 vol. of cold absolute ethanol and washed with aoetone. The precipitate was dried in a dessicator at 4°C. For crystallization, the dried pellet was dissolved in appropriate solutions. Samples (20 to 200 4) were placed in the depressions of a Pyrex spot-plate which rested on a platform made from an inverted half of a Petri dish. This was placed in a clear plastic box to which was added a 25-ml reservoir of isopropanol/water or 2-methyl-2, 4-pentanediollwater. Crystallization then occurs over a variable time period by vapor phase equilibrium at 4°C. (i) Orthorhornbic crystals Orthorhombic crystals were obtained from crystallization solutions containing 2 to 30 mg tRNA/ml, 10 to 80 mM-cacodylate buffer (pH 5-O to 7-O), 10 to 80 ma6-MgCl, and 40 mM1 to 6 mna-spermine hydrochloride. Optimum conditions are 15 mg tRNA/ml, cacodylate buffer (pH 6-O), 40 mM-MgC& and 3 mM-spermine hydrochloride. A reservoir of 9% isopropanol was generally used; however, a reservoir of 10% pentanediol with 4% pentanediol in the crystallization solution can also be used. (ii) Hexagonal crystals Hexagonal crystals were grown by using the same crystallizing solution described in section (i) above (optimum conditions) but with a reservoir of 4% isopropanol. Hexagonal crystals were also formed preferentially either by removal of spermine or by the addition of 1 ma-CoCl, or 2 m&r-tetraphenylarsonium chloride to the standard solution. (iii) Cubic crystals Cubic crystals have been obtained under a variety of conditions. They were grown reproducibly from a solution of 15 mg tRNA/ml, 40 mM-cacodylate buffer (pH 5*2), 40 mM-MgCl,, 3 InM of a thiolated analogue of spermine (2-[3-(4-aminobutyl-amino) propylamino] ethanethiol triphosphate) and 3 m-mercuric acetate with a reservoir of 9% isopropanol. Cubic crystals were also obtained with conditions similar to those used for orthorhombic crystals but with the addition of 40 mM-EDTA. On rare occasions these crystals were obtained under conditions identical to those used for orthorhombic crystals. (iv) Monoclinic crystals In a number of experiments, various polyamines were used in place of spermine. Monoclinic crystals developed in solutions containing 10 mg tRNA/ml, 40 mlvr-cacodylate buffer (pH 6.0), 40 mrd-magnesium acetate and 3 rnM of the thiolated spermine analogue cited above, These crystals began to appear after 1 to 2 days at 4°C and grew as clusters. (b) X-ray data collection The crystals were examined by mounting them, with a microdroplet of mother liquor, in glass capillaries which had been previously equilibrated with the crystallization buffer. Precession photographs were obtained using an Elliot rotating snode X-ray generator fitted with a copper target. The photographs were exposed from 12 to 24 h, during which time the temperature of the crystal was maintained at 8’C. Intensity data was obtained by processing the precession fllms on an optical drum scanner. Diffractometer data was collected using a modified Picker FAGS-1 system having a crystal-to-oounter distance of

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520 mm. Helium tubes were used in the paths of the incident and diffracted X-ray beams and the crystal temperature was maintained at 8°C. Intensity data were collected using an omega step scan technique.

3. Results Plate I shows photomicrographs of four of the crystal forms described in this paper. The crystal forms are typical and readily differentiated. Plate II shows diffraction patterns produced by these different crystals. The crystallographic parameters associated with the different crystal forms are given in Table 1, which also lists the effective resolution of the various diffraction patterns. The intensity fall-off in the diffraction photographs (Plate II) can be described quantitatively by plotting the averaged intensity vers~.~resolution, as shown in Figure 1. These curves were obtained from precession photographs. They represent projection data only,

A FIU. 1. Average value of the diffraction intensity patterns shown in Plate II. Curve A, orthorhombic;

as a function of resolution for the diffraction curve B, hexagonal; curve C, cubic; curve D,

(a) Orthorhombic crystals Crystals of tRNA in the space group P2,22, are shown in Plate I(a). These crystals grow preferentially in the a direction and always have one short dimension parallel to the c axis. When orthorhombic crystals are grown under optimum conditions, using a 9% isopropanol reservoir, an unusual sequence of events is observed. Within the first 12 hours, the crystallizing solution is filled with a large number of hexagonal crystals, less than 50 pm in diameter. After a few days these hexagonal crystals begin to disappear and clusters of orthorhombic plates begin to grow. These plates continue to grow for two to three weeks while the hexagonal crystals disappear. The orthorhombit crystals are in the form of long flat plates, 1000 to 2000 pm long and 200 to 600 pm wide. The thickness is the most critical dimension and commonly reaches 80 to 15Opm. Crystals of this size occurred in approximately 95% of the crystallization attempts. The second orthorhombic crystal form in Table 1, P2,2,2,, was only observed once, when KAuCl, wits added to preformed crystals. It is not known whether the auric chloride induced a transformation from P2,22, to P2,2,2, or whether the crystals were

P6,22 P6122

R32§

I4,32

Hexagonal

Trigonal

Cubic

C222f $

154

126

82 104

60.6

154

7 7 3

6 1

416

t Isopropanol. j: 2-methyl-2, C-pentanediol. 3 Cramer et al. (1970). I/ a axis unique

365

MPD, t-butanol

8

1 1

137 181

236 193

154

IP Ip, MPD

2.3 6.5 15 1 2

1

19-5-29.8 14.5 121

29

p212121

110-161 104 234

63-56 48 85.0

30-33

P2,22,

Orthorhombic

IP

IP MPD, t-butanol

Ip, MPDf

IPY

4

1

Precipitant

11.7

Resolution (4

90

63

56

33

%I1

hfonoclinic

Unit cell

Molcoules per asymmetric unit

tRNA crystals

vol. x lo-4 A

~(4

in yeast phe~ylalanine

do)

b(A)

44

Cry&al system

Polymorphkn

TABLE 1

PLATES I-IV PLATE I. Photomicrographs of the typical habits of yeast phenylalanine tRNA crystals examined in this study. (a) Thin-lath clusters of orthorhombic crystals (P2,22,); (b) hexagonal prisms (P6a22); (c) octahedral cubic forms (14,32); (d) a cluster of striated thin laths of monoclinic crystals (PZ,). PLATE II. X-ray diffraction patterns to-film distance was 100 mm and CuKol h0Z p = 14”; (b) hexagonal crystal: hk0 crystal OkZ p = 12”. The reflections for 3 A resolution.

of the various crystal forms shown in Plate I. The crystalradiation was used in all cases. (a) Orthorhombic crystal: p = 10”; (c) cubic crystal: FrJO /J = 14”; (d) monoclinic orthorhombic and cubic crystal forms extend to beyond

[facingp.424

PLATE I (a)and(b)

PLATE

I (c) and (d)

3

PLATE III PLATE III. Composite h0Z photograph of an orthorhombic form of yeast phenylalanine tRSA shown after 2 different time periods in the X-ray beam. The box encloses the region of greatest, intensity change. Photograph of the initial pattern is on the left; the photograph taken 48 h later is on the right.

PLATE IV. tRNA crystals.

Stationary X-ray diffraction photograph of the cubic form of yeast phenylalanine The crystal was initially aligned such that the [ 1 lo] direction was parallel to the X-ray beam and subsequently rotated 13” 30 min about the [liO] direction. Diffuse meridional reflections in the 3 to 4 ip region suggest the presence of double-helical structure in the molecule.

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already P2,2,21 before the addition, Both P2,22, and P2,2,2* crystals have very similar a and b axes; however, the c axis of the P2,2,2, crystal is somewhat shortened. The diffraction data of the P21212, form extended to only 65 8. One of the unusual characteristics of the P2,22, crystal form is the fact that its high resolution diffraction pattern appears to be somewhat unstable to small changes in solvent composition and exposure to X-rays. The instability is manifest not as a general loss of resolution, but rather as alterations in the intensities of different reflections in the pattern. This is illustrated in a simple experiment. Five sets of sequential diffractometer data were collected to a resolution of 5.5 B over a period of one week. The data sets were scaled together using Wilson statistics and the scaled values of some selected structure factors are plotted as a function of time in Figure 2.

FIG. 2. Scaled values of selected structure factors from 5 data sets as a function of time. The data were oollected sequentially over a period of 1 week. The dashed line corresponds to the average structure factor before scaling and the dotted line shows the average structure factor after scaling.

The dashed line corresponds to the average structure factor before scaling and the dotted line corresponds to the average structure factor after scaling. Even after scaling, some intensities decrease very rapidly such as (4,0, S), (4, 52) and (0,8, 16) and others such as (3, 7,4) and (6,0, 1) increase significantly as a function of exposure time. We have found that approximately 15% of the intensities show large changes after scaling. The reflections are mainly localizedin a cone of reciprocal space around the a* axis. This effect is further illustrated in Plate III where changes in the (600) and (601) reflections are clearly visible. The photograph, taken after 48 hours in the X-ray beam, shows a clearly visible increase in (6, 0, 1) and several significant changes are also visible in the (701) line. Similar effects can also be induced by slight changes in the crystal environment such as the addition of ions or changes in solvent oomposition. Since these changes are of the same order of magnitude as those associated with isomorphous replacement, they pose difficulties in the interpretation of possible heavyatom derivatives. 28

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(b) Hexagoruzl crystals Hexagonal crystals appear either singly or in clusters of 8 to 15 crystals as shown in Plate I(b). They are frequently larger than the orthorhombic crystals (1000 to 2000 pm in length and 200 to 400 pm in diameter) and usually have well developed hexagonal faces but are much softer than the orthorhombic forms. We have found three different unit cells in two different space groups, as listed in Table 1. The hexagonal diffraction pattern shown in Plate II(b) is from the space group P6,22 with a = 104 A and c = 193 A. The resolution of the hexagonal crystals is much poorer than that found in the other crystal forms and reflections extend to only 7 or 8 A. Closely related to these are the trigonal crystals described by Cramer et al. (1970). The data for that crystal form are also included in Table 1. (c) Cubic crystals

A transformation of crystalline types, similar to that described in the case of the orthorhombic crystals, also occurs during the formation of cubic crystals. Within 12 hours after preparing the crystallization a large number of crystal clusters resembling bundles of wheat appear in the solution. These subsequently disappear as the cubic crystals form. The cubic crystals have the form of octahedra (Plate I(c)) and their diffraction pattern shows strong reflections at a spacing of 3.2 A along the h and k axes (Plate II(c)) which is characteristic of a double-helical structure. The tilt photograph in Plate IV shows other strong reflections at spacings between 3.0 and 3.6 A. (d) Monoclinic

crystals

Examples of monoclinic crystals are shown in Plate I(d) and their corresponding diffraction pattern is shown in Plate II(d). The crystals do not have well-formed faces and usually appear striated under microscopic examination. They grow as clusters and take the form of elongated plates of dimensions 500 to 1000 pm by 100 to 300 pm by 30 to 60 Fm. The unit cell parameters are listed in Table 1. The a and b axes are similar to those of the orthorhombic crystals, but the c axis is less than half as long. This cell is similar to the monoclinic form of yeast phenylalanine tRNA recently reported by Iohikawa & Sundaralingham (1972), which was obtained under conditions identical to those we use for the orthorhombic form. As can be seen from Plate II, the resolution of the monoclinic crystals is better than that of the hexagonal form but less than that of the orthorhombic form.

4. Discussion One of the interesting features of yeast phenylalanine tRNA is its high degree of polymorphism. This behavior is compatible with a molecule whose surface is largely repetitive and, in particular, with a molecule whose surface is densely populated with charged acidic groups. Interactions between molecules of this type should be sensitive to the type and concentration of cations available for charge neutralization: divalent cations such as Mg2 + and Ca2+ result in non-stereospecific interactions between charged regions on the molecules while cations such as Co2+ and Mn2+ may produce more specific interactions due to their stronger anisotropic ligand-binding

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abilities. Complicated organio cations suoh as spermine and spermidine should also produce specific interactions as a result of their anisotropic charge distribution. It is interesting to note that apparently the only high-resolution forms of crystalline yeast Phe-tRNA that have been obtained have contained spermine or a related compound in the crystallization medium. A curious feature in the preparation of P2,22, crystals is the transient formation of hexagonal crystals (P6,22) from which the orthorhombic form then grows. This phenomenon can be explained by the assumption that the orthorhombic lattice is more stable than the hexagonal lattice but that it is able to nucleate much less readily. Even though the orthorhombic lattice appears to be more stable than the hexagonal lattice, diffraction from the orthorhombic crystals is still sensitive to changes in the crystallization buffer and exposure to X-rays. The changes which occur are localized in a cone about the a* axis and, therefore, are due to a change in the molecular paoking along the a direction or to a very specific intramolecular movement whose major component is in the a direction. In view of the high degree of polymorphism of this species of tRNA, a change in molecular packing seems plausible. If, on the other hand, the changes are due to a specific intramolecular movement, implying the existence of two closely related, stable conformations of the tRNA molecule, this could be important in the interpretation of the role of tRNA in protein synthesis. These changes have only been observed in the P2,22, lattice; however, their presence has not been sytematically investigated in the other lattices. A preliminary investigation of the monoclinic lattice suggests that it is more stable than the P2,22, form. This may be due to the way in which the molecules are packed in the two lattices which by symmetry must be head-to-head and tail-to-tail in the orthorhombic cell and head-to-tail and tail-to-head in the monoclinic cell. Plots of average intensity as a function of resolution have been reported for numerous protein crystals and similarities in the positions of the maxima have been interpreted in terms of tertiary structural features (Rossmann et al., 1967). In particular, peaks commonly occurring at 10 A and 4-5 A resolution were taken as indicative of the a-helical content of the protein and the interpeptide vectors, respectively. Although the data shown in Figure 1 represents only single sections in reciprocal space for each crystal form, they suggest that similar interpretations might be made if the analysis were carried to three dimensions. The appearance of peaks near 7.5 A and 5 A for the various forms could be indicative of tertiary structural characteristics of the tRNA molecule. The reflections in the 3.2 A region in the cubic lattice photograph (Plate II(c)) and the reflections between 3.0 A and 3.6 A in the tilted cubic lattice photograph (Plate IV) probably arise from double-helical segments in the tRNA molecule. It has been pointed out previously that the diffraction pattern produced by the orthorhombic crystal form shows characteristics of double-helical segments parallel to the long axis of the molecules (Kim et al., 1971). It is therefore likely that the long axis of the molecules in the cubic cell is orientated along the three 4-fold axes. Tilted hexagonal crystal photographs show no 3 A reflections in contrast to the cubic and orthorhombic crystal forms. Based on packing considerations, however, the long axis of the tRNA molecules should be parallel to the 6-fold hexagonal axis. The absence of strong 3 A reflections in this crystal form is probably due to the large degree of internal disorder, as indicated by the low resolution of the hexagonal diffraction patterns. 28’

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We thank Drs James C. Powers, John A. Montgomery and P. G&n Lenhert for assistance. We would also like to thank Carol Craig for the preparation of this manuscript. This work was supported by research grants from the National Institutes of Health, the National Science Foundation, the National Aeronautics and Space Administration, the American Cancer Society and the Damon Rnnyon Found&ion. REFERENCES Blake, R. D., Fresco, J. R. & Langridge, R. (1970). Nature, 225, 32. Cramer, F., von der Haar, F., Holmes, K. C., Saenger, W., Schlimme, E. & Schulz, G. E. (1970). J. Mol. Bid. 51, 523. Icbikawa, T. & Sundar~lingham, M. (1972). Nature, 236, 174 Kim, S. H., Quigley, G., Suddath, F. L. & Rich, A. (1971). Proc. Nat. Acud. I%., Wash. 68, 841.

Mirsabekov, A. D., Rhodes, D., Finch, J. T., Klug, A. 237, 27. Rossmann, M. G., Jeffery, B. A., Main, P. & Warren, Wash. 57, 515. Schevitz, R. Mr., Navia, M. A., Bantz, D. A., Cornick, Sigler, P. B. (1972). Science, 177, 429. Young, J. D., Book, R. M., Nishimura, S., Ishiknra, H., Labrmsuskas, M. & Conners, P. G. (1969). Science,

& Clark, B. F. C. (1972). Nature, S. (1967). Proc. Nut. Acud. Sci., G., Rosa, J. J., Rosa, M. D. H. & Yamada, Y., RajBhandary, 166, 1527.

U. L.,