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Pf1 bacteriophage replication-assembly complex
J. Mol. Biol. (1983) 171, 229-232
Pfl Bacteriophage Replication-Assembly Complex X-ray Fibre Diffraction of the High Humidity Form The helical intracellular nucleoprotein complex of Pfl bacteriophage has been studied by X-ray fibre diffraction in various hydration states. The helix pitch changes from 44 A in dry fibres to 55 A in wet fibres, whereas the unit rise between subunits in the helix apparently does not change with humidity. This result indicates that the nucleoprotein assembly twists more readily than it stretches. This is consistent with its biological role of tightening the viral DNA into a more compact form for packaging in the virion.
The gene 5 protein of filamentous bacteriophages binds to newly made singlestranded intracellular DNA. Several thousand gene 5 protein molecules combine with one DNA molecule to form a helical nucleoprotein complex about 1 #m long by 90 A diameter. The DNA in the complex is topologically a circular, single strand stretching down the length of the complex, with each of the two antiparallel DNA strands binding one gene 5 protein molecule every four nucleotides. No base-pairing between the DNA strands has been observed; instead, the two antiparailel nucleoprotein strands are held together in the complex by dimerization between gene 5 protein molecules on opposite strands. The helix followed by the complex is flexible and open, with the two nucleoprotein strands forming one quite wide groove and no discernible second groove. There are some differences between the complexes of the two species of filamentous bacteriophage that have been studied in detail. For the P f l species, Mr = 15,400 for the gene 5 protein, and the helix pitch of the complex is about 50 A; for the fd species, Mr = 9690 and the pitch is about 100 A. During assembly of the virion, the gene 5 protein is displaced from the DNA by the gene 8 protein (the major coat protein). This displacement is associated with a tightening of the DNA helix involving several hundred rotations of one end of the DNA with respect to the other end. The P f l complex gives better X-ray diffraction patterns than the fd complex, and has therefore been studied in more detail by structural techniques (Gray et al., 1982; Kneale & Marvin, 1982; Kneale et al., 1982). X-ray fibre diffraction patterns of the P f l complex at low and high humidity are shown in Figure I. The outer parts of the two patterns (beyond about 0-1 A-1) are generally similar, indicating that the subunit shape does not change significantly with humidity. The inner parts of the two patterns differ in several respects, indicating that the relation between subunits in the assembly does change with humidity. In particular, the pitch defined by the first three layerlines changes from 44 A at low humidity (Kneale et al., 1982) to 55 A at high humidity; and the second and third layer-lines associated with the J2 and J3 Bessel function contributions are significantly less intense in the low humidity 0022-2836/83/340229-04 $03.00/0
229
© 1983 AcademicPress Inc. (London)Ltd.
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form than in the high humidity form, as expected if there is a wider groove at high humidity. The fractional change in pitch with changing hydration is as great as the fractional change in a, the centre-to-centre distance between particles (Fig. 2). This implies that the interaction between adjacent helical turns along the helix is weak, and the dominant forces stabilizing the helix are in the contacts between adjacent subunits along the helix. One feature of the outer part of the diffraction pattern that does not change with humidity is the spacing of the strong intensity in the 7"5 A meridional region, which was attributed to the unit rise between subunits in the analysis of the low humidity pattern (Kneale et al., 1982). Attributing the corresponding region of intensity on the high humidity pattern to the unit rise, when taken together with the observed change in helix pitch between the two humidities, implies that the unit twist between subunits changes from 61 ° at low humidity to 48 ° at high humidity (or the number of units per turn of ;he helix changes from 5-9 to 7.5). Attributing this change in helix pitch to a change in unit twist rather than a change in unit rise implies that the length of the complex does not change significantly in the fibre. In contrast, the length of the complex as seen in the electron microscope depends substantially on the method of preparation (Gray et al., 1982; Kneale et al., 1982). This distinction need not concern us here, in view of the rather different environments of the complex on an electron microscope grid and in a fibre.
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FIG. 2. Variation in helix pitch P (filled symbols) and strong meridional spacing dm (open symbols) with humidity. Since the nominal relative humidity is not a precise measure of water content of the fibre, the measurements are plotted against the unit cell dimension a between the particles, as described for fd virions (Marvin, 196fi). Measurements were made on 3 different fibres, indicated by the 3 different symbols.
232
G. G. KNEALE AND D. A. MARVIN
An alternative interpretation of these changes in the diffraction pattern is t h a t the intensity in the 7.5 A meridional region should not be attributed to a Jo Bessel function contribution at high humidity. Instead, this intensity at high humidity might be due to a J1, by analogy with the humidity-induced transition between the B and C forms of DNA (Marvin et al., 1961), In this case, one might expect a discontinuity in the spacing of the 7.5 A meridional region as it moves from Jo to J1 with increasing humidity. In fact, no such discontinuity is observed (Fig. 2). Furthermore, the angular breadth of the 7.5 .A intensity is consistent with a J0, given the disorientation in the fibre (11.5°), but is less t h a n expected for two J1 Bessel function contributions centred on either side of the meridian and overlapping across the meridian. Although the experiments are not conclusive, we favour the first interpretation, t h a t the unit twist and not the unit rise of the complex changes with humidity. This interpretation is consistent with the biological function of the complex, which must promote a substantial change in unit twist as the DNA passes from the complex to the virion. European Molecular Biology Laboratory Heidelberg, Federal Republic of Germany
G. G. KNEALE t D. A. MARVIN$
Received 15 August 1983 REFERENCES Gray, C. W., Kneale, G. G., Leonard, K. R., Siegrist, H. & Marvin, D. A. (1982). Virology, 116, 40-52. Kneale, G. G. & Marvin, D. A. (1982). Virology, 115, 53-60. Kneale, G. G., Freeman, R. & Marvin, D. A. {1982). J. Mol. Biol. 155, 279-292. Marvin, D. A. (1966). J. Mol. Biol. 15, 8-17. Marvin, D. A. & Nave, C. {1982). In Structural Molecular Biology (Davies, D. B., Saenger, W. & Danyluk, S., eds), pp. 3-44, Plenum Press, New York. Marvin, D. A., Spencer, M., Wilkins, M. H. F. & Hamilton, L. D. (1961). J. Mol. Biol. 3, 547-565. Edited by A. Klug
t Author to whom correspondence should be sent. Present address: Department of Chemistry, University of Cambridge, Cambridge, England. :~Present address: Department of Biochemistry,University of Cambridge, Cambridge, England.
Pfl Bacteriophage Replication-Assembly Complex X-ray Fibre Diffraction of the High Humidity Form The helical intracellular nucleoprotein complex of Pfl bacteriophage has been studied by X-ray fibre diffraction in various hydration states. The helix pitch changes from 44 A in dry fibres to 55 A in wet fibres, whereas the unit rise between subunits in the helix apparently does not change with humidity. This result indicates that the nucleoprotein assembly twists more readily than it stretches. This is consistent with its biological role of tightening the viral DNA into a more compact form for packaging in the virion.
The gene 5 protein of filamentous bacteriophages binds to newly made singlestranded intracellular DNA. Several thousand gene 5 protein molecules combine with one DNA molecule to form a helical nucleoprotein complex about 1 #m long by 90 A diameter. The DNA in the complex is topologically a circular, single strand stretching down the length of the complex, with each of the two antiparallel DNA strands binding one gene 5 protein molecule every four nucleotides. No base-pairing between the DNA strands has been observed; instead, the two antiparailel nucleoprotein strands are held together in the complex by dimerization between gene 5 protein molecules on opposite strands. The helix followed by the complex is flexible and open, with the two nucleoprotein strands forming one quite wide groove and no discernible second groove. There are some differences between the complexes of the two species of filamentous bacteriophage that have been studied in detail. For the P f l species, Mr = 15,400 for the gene 5 protein, and the helix pitch of the complex is about 50 A; for the fd species, Mr = 9690 and the pitch is about 100 A. During assembly of the virion, the gene 5 protein is displaced from the DNA by the gene 8 protein (the major coat protein). This displacement is associated with a tightening of the DNA helix involving several hundred rotations of one end of the DNA with respect to the other end. The P f l complex gives better X-ray diffraction patterns than the fd complex, and has therefore been studied in more detail by structural techniques (Gray et al., 1982; Kneale & Marvin, 1982; Kneale et al., 1982). X-ray fibre diffraction patterns of the P f l complex at low and high humidity are shown in Figure I. The outer parts of the two patterns (beyond about 0-1 A-1) are generally similar, indicating that the subunit shape does not change significantly with humidity. The inner parts of the two patterns differ in several respects, indicating that the relation between subunits in the assembly does change with humidity. In particular, the pitch defined by the first three layerlines changes from 44 A at low humidity (Kneale et al., 1982) to 55 A at high humidity; and the second and third layer-lines associated with the J2 and J3 Bessel function contributions are significantly less intense in the low humidity 0022-2836/83/340229-04 $03.00/0
229
© 1983 AcademicPress Inc. (London)Ltd.
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L E T T E R S TO T H E E D I T O R
231
form than in the high humidity form, as expected if there is a wider groove at high humidity. The fractional change in pitch with changing hydration is as great as the fractional change in a, the centre-to-centre distance between particles (Fig. 2). This implies that the interaction between adjacent helical turns along the helix is weak, and the dominant forces stabilizing the helix are in the contacts between adjacent subunits along the helix. One feature of the outer part of the diffraction pattern that does not change with humidity is the spacing of the strong intensity in the 7"5 A meridional region, which was attributed to the unit rise between subunits in the analysis of the low humidity pattern (Kneale et al., 1982). Attributing the corresponding region of intensity on the high humidity pattern to the unit rise, when taken together with the observed change in helix pitch between the two humidities, implies that the unit twist between subunits changes from 61 ° at low humidity to 48 ° at high humidity (or the number of units per turn of ;he helix changes from 5-9 to 7.5). Attributing this change in helix pitch to a change in unit twist rather than a change in unit rise implies that the length of the complex does not change significantly in the fibre. In contrast, the length of the complex as seen in the electron microscope depends substantially on the method of preparation (Gray et al., 1982; Kneale et al., 1982). This distinction need not concern us here, in view of the rather different environments of the complex on an electron microscope grid and in a fibre.
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Unit cell dimension, a (~)
FIG. 2. Variation in helix pitch P (filled symbols) and strong meridional spacing dm (open symbols) with humidity. Since the nominal relative humidity is not a precise measure of water content of the fibre, the measurements are plotted against the unit cell dimension a between the particles, as described for fd virions (Marvin, 196fi). Measurements were made on 3 different fibres, indicated by the 3 different symbols.
232
G. G. KNEALE AND D. A. MARVIN
An alternative interpretation of these changes in the diffraction pattern is t h a t the intensity in the 7.5 A meridional region should not be attributed to a Jo Bessel function contribution at high humidity. Instead, this intensity at high humidity might be due to a J1, by analogy with the humidity-induced transition between the B and C forms of DNA (Marvin et al., 1961), In this case, one might expect a discontinuity in the spacing of the 7.5 A meridional region as it moves from Jo to J1 with increasing humidity. In fact, no such discontinuity is observed (Fig. 2). Furthermore, the angular breadth of the 7.5 .A intensity is consistent with a J0, given the disorientation in the fibre (11.5°), but is less t h a n expected for two J1 Bessel function contributions centred on either side of the meridian and overlapping across the meridian. Although the experiments are not conclusive, we favour the first interpretation, t h a t the unit twist and not the unit rise of the complex changes with humidity. This interpretation is consistent with the biological function of the complex, which must promote a substantial change in unit twist as the DNA passes from the complex to the virion. European Molecular Biology Laboratory Heidelberg, Federal Republic of Germany
G. G. KNEALE t D. A. MARVIN$
Received 15 August 1983 REFERENCES Gray, C. W., Kneale, G. G., Leonard, K. R., Siegrist, H. & Marvin, D. A. (1982). Virology, 116, 40-52. Kneale, G. G. & Marvin, D. A. (1982). Virology, 115, 53-60. Kneale, G. G., Freeman, R. & Marvin, D. A. {1982). J. Mol. Biol. 155, 279-292. Marvin, D. A. (1966). J. Mol. Biol. 15, 8-17. Marvin, D. A. & Nave, C. {1982). In Structural Molecular Biology (Davies, D. B., Saenger, W. & Danyluk, S., eds), pp. 3-44, Plenum Press, New York. Marvin, D. A., Spencer, M., Wilkins, M. H. F. & Hamilton, L. D. (1961). J. Mol. Biol. 3, 547-565. Edited by A. Klug
t Author to whom correspondence should be sent. Present address: Department of Chemistry, University of Cambridge, Cambridge, England. :~Present address: Department of Biochemistry,University of Cambridge, Cambridge, England.