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X-ray diffraction studies of the RNA tetramer GpGpCpUp
.J. Mol.
X-ray
Rid.
(1981) 148, 103-106
Diffraction
Studies of the RNA
Tetramer
GpGpCpUp
The synthetic RNA tetramer GpGpCpUp has been studied by X-ray diffraction of single crystals and fibres. A preliminary crystallographic analysis of single crystals implies that two GpGpCpUp strands form a short antiparallel double helix with (:. U base-pairs at the ends of it. As diffraction intensities of single crystals fall into decay, the diffraction pattern gradually changes into a fibre pattern similar to that of .-I-RNA or of the rl form of DNA.
The RNA fragment GpGpCpUp (hereafter to be termed GGCU) is not a complete self-complementary tetramer, but expected to form a short antiparallel double helix containing terminal C:. U base-pairs. The existence of G. U base-pairs was for codon-anticodon initially proposed by Crick (1966) in his wobble hypothesis interaction. This base-pair has since been included very often both within and at the end of the helical stems of the transfer RNA cloverleaf model. Direct evidence for the presence of a G. U base-pair has been provided, for the first time, by the Xat 2.5 A resolution which has been ray crystal structure of yeast tRNAPh’ established for both the monoclinic (Ladner et al., 1975; Stout et al., 1976) and orthorhombic (Quigley et al., 1975; Sussman & Kim, 1976) polymorphic forms. These structures have revealed that the G .U opposition is involved in the wobble base-pair as predicted by Crick (1966). M izuno & Sundaralingam (1978) have found that’ the G U base-pairing within the amino acid stem of yeast tRNAPh’ leads to remarkably different base stacking with the Watson-Crick base-pairs situated on either sides of it : the G. U base-pair exhibits greater stacking interactions with the Watson-Crick base-pair following it on the 3’.side of 0 than the Watson-Crick base-pair preceding it on the 5’.side of G. This observation has important implications on the occurrence and the stability of the C . U base-pair at the end of a helical stem. Indeed, at the ends of helical stems of known tRNAs, the G. U basepair type which shows greater stacking with the adjacent base-pair is overwhelmingly distributed over the alternative G .U base-pair type (Mizuno & Sundaralingam, 1978). In this study, the GGCU tetramer was chosen. because its sequence corresponds to the former type G. U base-pair when it forms an antiparallel double helix by self-association. Thus, X-ray diffraction studies of the (ZCU could provide an approach to the goal of determining the structure of and around a G. U base-pair at atomic resolution. The sodium salt of the GGCU was synthesized using the phosphotriester method (Ohtsuka et al.. 1979). Crystallization was carried out in the cold room (4 to 6°C) by vapour diffusion from a solution of 15 miv-sodium cacodylate buffer (pH 6-O). 5 mm-spermine tetrachloride, 7,5 mM-MgCl, and 2.3 mM sodium salt of the GGCC tetramer. Transparent crystals appeared within 24 hours as rod-shape or rectangular plates that grow up to dimensions of 1.5 mm x 04 mm x 0.2 mm. X002~2836/81j1301034
$02.00/O
103 ic‘, 1981 Acitdemic.
Press Inc.
(London)
Ltd.
104
H.
MIZYhJO,*
E’/’
AL.
ray data were measured by diffractometer. The crystals are tetragonal with space group P42,2 and with a = b = 3957 and t = 32.75 A, so that there would be two GGCU fragments in a crystallographic asymmetric unit (see below). The cell dimensions were compared with those of the DNA double-helical fragment d(CpGpCpG) crystal whose structure has recently been solved (Drew et al.. 1980: Crawford et al., 1980). The present a and b dimensions correspond to about double the length of the a axis (19.50 A) of the dCGCG, while the present c dimension corresponds to about half the length of the c axis (64.67 A) of the dCGCU. The unit cells are of similar volume and so therefore also are the volumes of the asymmetric units. This simi1arit.y may reflect that one cylindrical duplex containing two GCXU strands could pack in an asymmetric unit as does the dCGCG, although the dCG(‘(: crystallizes as a left-handed duplex which is originally found in the crystal structure of the DNA hexamer d(CpGpCpGpCpG) (Wang rt al., 1979). It should be noted that the cylindrical representation of the GGCU molecules is consistent with the presence of G .17 base-pairs at both ends of the double helix composed of an antiparallel organization of the two GGCTT strands. Judging from the cylindrical dimension, the GGCI? presumably form an extended and slim helix like Z-DNA found in the dCGCGCG or like Z’-DNA in the dCGCG. The packing diagram is in contrast to that found in the CGCG crystal (Fig. 1). The square (or tetragonal) arrangement of cylindrical molecules can hardly form so close a packing as the hexagonal one. The formation of such a square packing of GGCU molecules is mainly due to the additional phosphates at the terminal U residues together with counter cations bound to them. Neighbour molecules along the c axis are considered to be related by a diagonal crystallographic dyad axis, forming a model with eight base-pairs up the c axis. The paired base arrangement appears related to the (6 1 8) reflection with a spacing of 3.46 A. because this reflection is observed to be
H 39.57
a
FIG. 1. (left) Projection on the ab plane showing inferred packing of the GpGpCpUp double helices. The space group is P42,2. Each circle represents cylindrical double helices. and contains 2 helices in the c axis repeat of 32.75 A. (right) Projection on t,he a6 plane showing packing of the d(CpGpCpG) double helices (Drew et al.. 1980; Crawford et al.. 1980). The space group is c7222,. Each circle contains 4 helices in the e axis repeat of 64.67 A.
LETTERS
TO
THE
EDITOR
105
the strongest except for the (2 0 0) reflection. Therefore, the base-pair planes are expected to lie nearly or roughly on the (6 1 8) planes which are tilted 32.2” from perpendicular to the c axis. Helix axes should also be tilted from the c axis ; if the helix axes were parallel to the c axis, the rise per base is too long (499 A) and basepair tilt is too large (32.2”), while, if helix axes were perpendicular to the (6 1 8) plane, base-pair stacking between molecules would result in poor interactions or in unacceptable geometry. Helix axes are therefore considered to be tilted from the c axis so as to make a compromise between the above cases. Tt should be noted that as diffraction intensities of transparent single crystals fall into decay, crystals turn whitish, and the diffraction pattern gradually changes into a fibre pattern (Fig. 2). The intensity distribution of a series of strong reflections on the equatorial, the 1st and the 2nd layer-lines and of weak reflections on the 3rd layer-line is generally similar to the pattern given by A-RNA or by the =1 form of DNA. While the intensity distribution of near-meridional diffraction on the 6th, the 7th, the 8th and the 9th layer-lines is similar to the corresponding distribution of the A form of DNA rather than of A-RNA. The axial repeat is markedly shrunk to 25.8 A from the c axis (32.75 A) of the single crystal. Some fibre pattern photographs taken at different angles from perpendicular to the X-ray beam allow meridional diffraction on the 10th layer-line. This implies a IO-fold helix with a repeat distance of 25-S/10 = 2.58 A. These helical parameters are ver? similar to those deduced from the crystal structure of GpC rather than those from the crystal structure of ApU (Rosenberg rt al., 1976).
FIG. 2. A 5” precession photograph (without layer-line screen) GpGpCpUp. Film distance is 60 mm, and the c axis is vertical.
of a paracrystalline
specimen
of the
H. MIZlJNO h'7'.4L
106
As is partly seen in Figure 2, reflections arising from the single crystal can be observed to be overlapped on the fibre pattern. This reflects that a single crystal phase and a polymeric phase exist as a mixture. The phase transition might occur gradually. The GGCU tetramer molecules in the crystal turn out to be a continuous lo-fold structure in which the tetramers are conformationally linked in the helical direction without being interconnected. Detailed structural models in both phases will be found to account for the differences in the contacts between neighbouring double helices along the helix axis (where G. U base-pairs could stack together) in crystals and in fibres, and might be of potential use in understanding the actual nature of a stereochemically plausible transition. Further information from the fibre pattern is being gathered. Complete single crystal three-dimensional data have been collected to a resolution of 1.1 !I using a Rigaku diffractometer. Structural analysis at atomic resolution is in progress. The authors thank Dr Takaji Fujiwara for Kensaku Hamada for their technical assistance.
useful
discussions,
and Keizo
Ogawa
Laboratory of Physical Chemistry Faculty of Pharmaceutical Sciences Osaka University, Suita, Osaka 565, Japan
HIKOSHI MIZL~NO KEN-ICHI TOMITA
Laboratory
EIKO NAKACAWA E~rco OHTSVKA Morzro IICEHARA
of Organic Chemistry
Faculty of Pharmaceutical Osaka University, Suita, Received
24 November
Sciences Osaka 565, Japan
and
1980
REFERENCES Crawford, J. L., Kolpak, F. J., Wang, A. H.-J., Quigley, G. ,I., van Boom, J. H., van der Marel, G. & Rich, A. (1980). Proc. Sat. Acad. Sci., U.S.A. 77, 401&4020. Crick, F. H. C. (1966). J. Mol. Biol. 19, 54t3555. Drew, H., Takano, T., Tanaka, S., Itakura, K. & Dickerson, R. E. (1980). Saturv (Lon>don). 286, 567-573. Ladner, J. E., Jack, A., Robertus, J. D., Brown, R. S., Rhodes, D., Clark, B. F. C. & Klug, A. (1975). Nml. Acids Res. 2, 1629-1637. Mizuno, H. & Sundaralingam, M. (1978). A’ucZ. Acids Res. 5, 4451-4461. Ohtsuka, E., Tanaka. T. & Ikehara, M. (1979). J. Amer. Chem. Sot. 101, 640%6414. Quigley, G. J., Seeman, N. C., Wang, A. H.-J., Suddath, F. L. & Rich, A. (1975). Sucl. Acids Res. 2, 2329-2339. Rosenberg, J. M., Seaman, N. C., Day, R. 0. & Rich, A. (1976). Biochem. Biophya. Res. Commun. 69, 979987. Stout, C. D., Mizuno, H., Rubin, J., Brennan: T., Rao, S. T. & Sundaralingam. M. (1976). n’ucl. Acids Res. 3, 1111-1123. Sussman, cJ. L. & Kim, S. H. (1976). Biochem. Biophys. Res. Commun. 68, 89-96. Wang, A. H.-J., Quigley, G. ,J., Kolpak, F. J., Crawford, J. L., van Boom, J. H., van der Marel, G. & Rich, A. (1979). Nature (London), 282, 680-686.
X-ray
Rid.
(1981) 148, 103-106
Diffraction
Studies of the RNA
Tetramer
GpGpCpUp
The synthetic RNA tetramer GpGpCpUp has been studied by X-ray diffraction of single crystals and fibres. A preliminary crystallographic analysis of single crystals implies that two GpGpCpUp strands form a short antiparallel double helix with (:. U base-pairs at the ends of it. As diffraction intensities of single crystals fall into decay, the diffraction pattern gradually changes into a fibre pattern similar to that of .-I-RNA or of the rl form of DNA.
The RNA fragment GpGpCpUp (hereafter to be termed GGCU) is not a complete self-complementary tetramer, but expected to form a short antiparallel double helix containing terminal C:. U base-pairs. The existence of G. U base-pairs was for codon-anticodon initially proposed by Crick (1966) in his wobble hypothesis interaction. This base-pair has since been included very often both within and at the end of the helical stems of the transfer RNA cloverleaf model. Direct evidence for the presence of a G. U base-pair has been provided, for the first time, by the Xat 2.5 A resolution which has been ray crystal structure of yeast tRNAPh’ established for both the monoclinic (Ladner et al., 1975; Stout et al., 1976) and orthorhombic (Quigley et al., 1975; Sussman & Kim, 1976) polymorphic forms. These structures have revealed that the G .U opposition is involved in the wobble base-pair as predicted by Crick (1966). M izuno & Sundaralingam (1978) have found that’ the G U base-pairing within the amino acid stem of yeast tRNAPh’ leads to remarkably different base stacking with the Watson-Crick base-pairs situated on either sides of it : the G. U base-pair exhibits greater stacking interactions with the Watson-Crick base-pair following it on the 3’.side of 0 than the Watson-Crick base-pair preceding it on the 5’.side of G. This observation has important implications on the occurrence and the stability of the C . U base-pair at the end of a helical stem. Indeed, at the ends of helical stems of known tRNAs, the G. U basepair type which shows greater stacking with the adjacent base-pair is overwhelmingly distributed over the alternative G .U base-pair type (Mizuno & Sundaralingam, 1978). In this study, the GGCU tetramer was chosen. because its sequence corresponds to the former type G. U base-pair when it forms an antiparallel double helix by self-association. Thus, X-ray diffraction studies of the (ZCU could provide an approach to the goal of determining the structure of and around a G. U base-pair at atomic resolution. The sodium salt of the GGCU was synthesized using the phosphotriester method (Ohtsuka et al.. 1979). Crystallization was carried out in the cold room (4 to 6°C) by vapour diffusion from a solution of 15 miv-sodium cacodylate buffer (pH 6-O). 5 mm-spermine tetrachloride, 7,5 mM-MgCl, and 2.3 mM sodium salt of the GGCC tetramer. Transparent crystals appeared within 24 hours as rod-shape or rectangular plates that grow up to dimensions of 1.5 mm x 04 mm x 0.2 mm. X002~2836/81j1301034
$02.00/O
103 ic‘, 1981 Acitdemic.
Press Inc.
(London)
Ltd.
104
H.
MIZYhJO,*
E’/’
AL.
ray data were measured by diffractometer. The crystals are tetragonal with space group P42,2 and with a = b = 3957 and t = 32.75 A, so that there would be two GGCU fragments in a crystallographic asymmetric unit (see below). The cell dimensions were compared with those of the DNA double-helical fragment d(CpGpCpG) crystal whose structure has recently been solved (Drew et al.. 1980: Crawford et al., 1980). The present a and b dimensions correspond to about double the length of the a axis (19.50 A) of the dCGCG, while the present c dimension corresponds to about half the length of the c axis (64.67 A) of the dCGCU. The unit cells are of similar volume and so therefore also are the volumes of the asymmetric units. This simi1arit.y may reflect that one cylindrical duplex containing two GCXU strands could pack in an asymmetric unit as does the dCGCG, although the dCG(‘(: crystallizes as a left-handed duplex which is originally found in the crystal structure of the DNA hexamer d(CpGpCpGpCpG) (Wang rt al., 1979). It should be noted that the cylindrical representation of the GGCU molecules is consistent with the presence of G .17 base-pairs at both ends of the double helix composed of an antiparallel organization of the two GGCTT strands. Judging from the cylindrical dimension, the GGCI? presumably form an extended and slim helix like Z-DNA found in the dCGCGCG or like Z’-DNA in the dCGCG. The packing diagram is in contrast to that found in the CGCG crystal (Fig. 1). The square (or tetragonal) arrangement of cylindrical molecules can hardly form so close a packing as the hexagonal one. The formation of such a square packing of GGCU molecules is mainly due to the additional phosphates at the terminal U residues together with counter cations bound to them. Neighbour molecules along the c axis are considered to be related by a diagonal crystallographic dyad axis, forming a model with eight base-pairs up the c axis. The paired base arrangement appears related to the (6 1 8) reflection with a spacing of 3.46 A. because this reflection is observed to be
H 39.57
a
FIG. 1. (left) Projection on the ab plane showing inferred packing of the GpGpCpUp double helices. The space group is P42,2. Each circle represents cylindrical double helices. and contains 2 helices in the c axis repeat of 32.75 A. (right) Projection on t,he a6 plane showing packing of the d(CpGpCpG) double helices (Drew et al.. 1980; Crawford et al.. 1980). The space group is c7222,. Each circle contains 4 helices in the e axis repeat of 64.67 A.
LETTERS
TO
THE
EDITOR
105
the strongest except for the (2 0 0) reflection. Therefore, the base-pair planes are expected to lie nearly or roughly on the (6 1 8) planes which are tilted 32.2” from perpendicular to the c axis. Helix axes should also be tilted from the c axis ; if the helix axes were parallel to the c axis, the rise per base is too long (499 A) and basepair tilt is too large (32.2”), while, if helix axes were perpendicular to the (6 1 8) plane, base-pair stacking between molecules would result in poor interactions or in unacceptable geometry. Helix axes are therefore considered to be tilted from the c axis so as to make a compromise between the above cases. Tt should be noted that as diffraction intensities of transparent single crystals fall into decay, crystals turn whitish, and the diffraction pattern gradually changes into a fibre pattern (Fig. 2). The intensity distribution of a series of strong reflections on the equatorial, the 1st and the 2nd layer-lines and of weak reflections on the 3rd layer-line is generally similar to the pattern given by A-RNA or by the =1 form of DNA. While the intensity distribution of near-meridional diffraction on the 6th, the 7th, the 8th and the 9th layer-lines is similar to the corresponding distribution of the A form of DNA rather than of A-RNA. The axial repeat is markedly shrunk to 25.8 A from the c axis (32.75 A) of the single crystal. Some fibre pattern photographs taken at different angles from perpendicular to the X-ray beam allow meridional diffraction on the 10th layer-line. This implies a IO-fold helix with a repeat distance of 25-S/10 = 2.58 A. These helical parameters are ver? similar to those deduced from the crystal structure of GpC rather than those from the crystal structure of ApU (Rosenberg rt al., 1976).
FIG. 2. A 5” precession photograph (without layer-line screen) GpGpCpUp. Film distance is 60 mm, and the c axis is vertical.
of a paracrystalline
specimen
of the
H. MIZlJNO h'7'.4L
106
As is partly seen in Figure 2, reflections arising from the single crystal can be observed to be overlapped on the fibre pattern. This reflects that a single crystal phase and a polymeric phase exist as a mixture. The phase transition might occur gradually. The GGCU tetramer molecules in the crystal turn out to be a continuous lo-fold structure in which the tetramers are conformationally linked in the helical direction without being interconnected. Detailed structural models in both phases will be found to account for the differences in the contacts between neighbouring double helices along the helix axis (where G. U base-pairs could stack together) in crystals and in fibres, and might be of potential use in understanding the actual nature of a stereochemically plausible transition. Further information from the fibre pattern is being gathered. Complete single crystal three-dimensional data have been collected to a resolution of 1.1 !I using a Rigaku diffractometer. Structural analysis at atomic resolution is in progress. The authors thank Dr Takaji Fujiwara for Kensaku Hamada for their technical assistance.
useful
discussions,
and Keizo
Ogawa
Laboratory of Physical Chemistry Faculty of Pharmaceutical Sciences Osaka University, Suita, Osaka 565, Japan
HIKOSHI MIZL~NO KEN-ICHI TOMITA
Laboratory
EIKO NAKACAWA E~rco OHTSVKA Morzro IICEHARA
of Organic Chemistry
Faculty of Pharmaceutical Osaka University, Suita, Received
24 November
Sciences Osaka 565, Japan
and
1980
REFERENCES Crawford, J. L., Kolpak, F. J., Wang, A. H.-J., Quigley, G. ,I., van Boom, J. H., van der Marel, G. & Rich, A. (1980). Proc. Sat. Acad. Sci., U.S.A. 77, 401&4020. Crick, F. H. C. (1966). J. Mol. Biol. 19, 54t3555. Drew, H., Takano, T., Tanaka, S., Itakura, K. & Dickerson, R. E. (1980). Saturv (Lon>don). 286, 567-573. Ladner, J. E., Jack, A., Robertus, J. D., Brown, R. S., Rhodes, D., Clark, B. F. C. & Klug, A. (1975). Nml. Acids Res. 2, 1629-1637. Mizuno, H. & Sundaralingam, M. (1978). A’ucZ. Acids Res. 5, 4451-4461. Ohtsuka, E., Tanaka. T. & Ikehara, M. (1979). J. Amer. Chem. Sot. 101, 640%6414. Quigley, G. J., Seeman, N. C., Wang, A. H.-J., Suddath, F. L. & Rich, A. (1975). Sucl. Acids Res. 2, 2329-2339. Rosenberg, J. M., Seaman, N. C., Day, R. 0. & Rich, A. (1976). Biochem. Biophya. Res. Commun. 69, 979987. Stout, C. D., Mizuno, H., Rubin, J., Brennan: T., Rao, S. T. & Sundaralingam. M. (1976). n’ucl. Acids Res. 3, 1111-1123. Sussman, cJ. L. & Kim, S. H. (1976). Biochem. Biophys. Res. Commun. 68, 89-96. Wang, A. H.-J., Quigley, G. ,J., Kolpak, F. J., Crawford, J. L., van Boom, J. H., van der Marel, G. & Rich, A. (1979). Nature (London), 282, 680-686.