- Email: [email protected]
Quantized incorporation of RNA during assembly of tobacco mosaic virus from protein disks
.I. Mol. Biol. (1978) 126, 877-882
Quantized Incorporation of RNA During Assembly of Tobacco Mosaic Virus from Protein Disks Reassembly of tobacco mosaic virus from the isolated RNA and protein, supplied as a disk preparation consisting of over 75% as the disk aggregate, has been followed by the protection of the RNA from nuclease digestion. The sizes of the RNA fragments were determined on agarose/acrylamide gels. During the f&t few minutes the protected RNA is found to be “quantized” into discrete lengths, differing on average by about 50 or 100 nucleotides, corresponding to one or two turns of the virus helix and strongly supporting the llypothesis that elongation in the major direction, towards the 5’-hydroxyl end, is occurring by the direct addition of protein disks. Protected RNA of the full length found in tobacco mosaic virus is visible within six minutes of starting reassembly, and by 30 minutes most of the RNA is fully protected.
Under conditions where assembly of tobacco mosaic virus occurs readily and rapidly from it,s isolated RNA and coat protein, the protein exists as a mixture of small aggregates, called A-protein, and larger disk aggregates. These disks consist of two rings. each of 17 protein subunits (Durham et al., 1971; Durham & Klug, 1971), and are essential for the nucleation of virus assembly (Butler & Klug, 1971). Moreover, it appeared from the kinetics of the growth that they might be acting directly as the protein source during the rod elongation, as well as during the nucleation (Butler. 1972). This latter hypothesis has, however, been the subject of considerable debate (for a review see Butler & Durham, 1977). A major cause of this controversy was the apparent topological complexity in adding a closed ring or disk onto the end of a rod from which the free RNA tail would be protruding. This had been recognized from the start but the preliminary data indicated that disks were used during the elongation (Butler & Klug, 1971). More recently, from the combination of our studies on the nucleation region of TMVTRNA (Zimmern & Wilson, 1976; Zimmern & Butler, 1977 ; Zimmern, 1977) and on the structure of the disk aggregate (Champness et al., 1976), we were able to propose a hypothesis for t’he nucleation which largely overcomes this difficulty (Butler et al.. 1976). This involves the insertion of a special hairpin loop of the RNA into the central hole of the disk, from where it can bind round into the open “jaws” between the two rings of protein. This interaction would cause the disk to dislocate into a short helix, with the RNA entrapped between the turns of protein. This mode of nucleation results in a growing helix with both RNA tails protruding from one end, one directly and the other after doubling back down the central hole of the rod. We have confirmed that the major tail (the 5’-hydroxyl) is doubled back and that this structure is important for rapid elongation of the particles (Butler et al., 1977), and the two tails at the one end have also been pictured on partially assembled rods (Lebeurier et aE., 1977). Given this structure, elongation can, like the nucleation, occur by insertion of a loop of RNA into the jaws of a disk from its central hole; only now instead of the t Abbreviation
used:
TMV,
tobacco
mosaic
virus. 877
1,022-~X:j6/7x/:~6OX77--06
$02.00/O
:C) 1978 Academic
Press
Inc.
(London)
Ltd.
878
P.
J.
G.
BUTLER
AND
G.
P.
LOMONOSSOFF
special nucleation loop it is a “travelling loop” formed as the RNA turns back down the central hole of the rod. This loop is self-perpetuating as it can be reformed as the RNA is used up, by pulling more of the 5’-tail up the central hole. Such a mechanism of elongation makes it simple to comprehend how a complete disk can add during the elongation but it is, however, still argued that this is too complex a process to occur (Fukuda et al., 1978). We now present evidence from a direct method to show the involvement of disks during elongation. If subunits are added directly from a disk, one would expect the RNA to be incorporated in lengths of about 50 nucleotides (3 nucleotides per subunit x 17 subunits) or 100 nucleotides at a time, as one or both rings interact. During the isolation of the nucleation region of TMV RNA, which was undertaken by reacting the RNA with small amounts of a disk preparation and subsequently removing the unprotected tails with nuclease (Zimmern & Butler, 1977), a rough banding pattern suggestive of such a step-wise addition had indeed been seen (Zimmern, 1977). We have non investigated this banding in more detail over a considerably greater length of the RNA, to ensure that it does not occur only during the init,ial steps which were already known to require disks. The measurement of the lengths of the protected RNA is used as a direct measure of the elongation. Reassembly was carried out at 20°C in sodium phosphate buffer (pH 7-O), ionic strength 0.1 M> with a protein disk preparation at 8 mg/ml and RNA at 0.2 mg/ml final concentration. Preparations were as previously described for both the protein (Durham, 1972) and the RNA (Zimmern, 1975). Elongation was stopped and the uncoated RNA removed by the addition of CaCl, and micrococcal nuclease to 1 mM and 40 units/ml, respect’ively. This concentration of Ca2+ alone was found to totally inhibit elongation in some preliminary experiments. After digestion at 20°C for the desired time, 1,2-di(2-aminoethoxy)-ethane-N,N,N’,Nf,-tetraacetic acid was added to 5 mM to complex the Ca2 + , and the rodlets pelleted at 55,000 revs/min and 5°C for five hours in an M.S.E. 10 x 10 ml titanium rotor. The rodlets were resuspended in lOOmMNaCl, 1 mM-EDTA, lOmr\l-Tris-chloride (pH7.5), and the RNA extracted as before and precipitated twice with ethanol to remove the phenol. RNA lengths were determined by electrophoresis in 0.5% agarose/2.5% polyacrylamide gels prepared and run according to Peacock & Dingman (1968), but) with 0.1% sodium dodecyl sulphate added to all buffers. Samples were denatured just’ prior to application to the gel by heating to 100°C in 50% (v/v) formamide containing 10 mM-EDTA. After electrophoresis, the gels were soaked for 30 minutes in water, stained with 2 mg ethidium bromide/l and briefly washed in water again before photographing under short wavelengt,h ultraviolet light through a red filter. A time course for the elongation is shown in Figure 1, and shows a clear banding pattern and the steady elongation. Full length RNA is first protected by six minutes, in agreement’ with our previous estimates of the maximum rate of growth (Butler, 1972; Butler & Finch, 1973), and within 30 minutes the strong band is full length and the amount of smaller material is very similar to that in the original TMV RNA sample, showing that the reaction is complete within this time. This is in contrast to t,he longer times apparently required with OM-strain RNA and protein (Fukuda et al., 1978), and suggests that results from this strain should not be compared directly with those from the commonly used Vulgare strain. The lengths were estimated for the bands by extrapolation from those of the Escherichia coli ribosomal RNAs, which were taken as 1580 nucleotides (Ehresman et al., 1975) and 3100 nucleotides (Branlant
LETTERS R
I
TO
THE 4
2
3
COUI‘BR
of elongation
EDITOR 6
X7!l IO
15
30
T
R
6400
1230
470
FIG.
1. Time
of protected
RNA.
out for the times indicated (in min) and the uncoated tails remowd I,> Reaf ISA mbly was carried micrococcal nuclease for 30 min. E. coli ribosomal RNA was used as a markcI dig&it “ll with (track TMV RNA preparation used for the elongation was also examinrvl RI and the original banding pattern, together with some approximate corrected lengths (SW (track T) . The regular is indicated on the left., broken bars indicating t,he positions of weak bands. tlisscus iSi In in text),
et al.: 1976), plotting the logarithm of the length against the mobility. Because of the long extrapolation and reliance on only two markers, these sizes will not be accurattb but, t,o a first approximation, they do showa spacing of approximately 50 nucleotides over the whole range from 300 to 1000. Later experiments with many more markers (Fig. 2) show that the shorter ribosomal RNA migrates rather slower than expected for its size. If allowance is made for this, the spacing becomes about 54 nucleotides over the range from 470 to 1230. The corrected sizes are shown in Figure 1, and this is justified by the obvious similarities between the band patterns in the separate experiments shown in Figures 1 and 2. One noticeable feature of the RNA lengths is the wide range of sizes present at an?time. Taken together with the build-up of certain bands, this suggests that secondary
880
P.
J.
G.
BUTLER
AND
G.
P.
LOMONOSSOFF
structure of the RNA may be delaying the elongation at these points, most probably by obstructing the pulling of the RNA tail up the central hole of the rod. A further feature is the loss of discrete bands by about ten minutes. This must be the result of a spreading of the lengths of rods present and is presumably due to the loss or accretion of single protein subunits to or from the A-protein present in the disk preparation, rather than a strict addition only of intact disks to the growing particles. The most likely place for this to occur is at the minor 3’-tail, which is not doubled back down the rod but protrudes directly. Despite the reports that the 3’-tail is not coated at all before completion of coating of the 5’-tail (Lebeurier et al., 1977 ; Fukuda et al., 1978), coating of this tail appears to be independent of the major Y-tail and to occur throughout elongation (Lomonossoff & Butler, 1979), albeit at a much slower rate than the major tail, and it may very well occur from A-protein rather than from disks, and so be unquantized and smear the pattern from the other end. In another experiment (Fig. Z), the lengths of the RNA fragments were moro accurately determined, by including markers of cowpea chlorotic mottle virus RNA (Bancroft, 1971) and rabbit globin messengerRNA (Proudfoot et al., 1977), and measuring the bands which fell within the range of the markers rather than extrapolating. Over the range from 1260to 2750 nucleotides the bands are mainly separated by steps of about 100 nucleotides (or sometimes200), with a few st,epsof about 50. Taking a weighted average over this range (to estimate the fundamental spacing) gives a step length of 99 nucleotides, with a standard deviation of &4%, in close agreement with the value of 102 nucleotides predicted for the length covered by the addition of complete disks. It is unlikely that the banding pattern is due to an artefact of the nucleasedigestion, such as unfavourable sequences.Micrococcal nuclease will cleave either single 01 double-stranded nucleic acids and is not very specific in sequencerequirement. The regular pattern of the band shows that such sequences,if they exist, would have to be repeated over a length of more than 1500 nucleotides, or roughly a quarter of the viral RNA. Moreover, no significant difference is visible in the patterns produced after 5 or 60 minutes digestion (Fig. 2, tracks a and b) showing that the pattern is not simply a result of underdigestion. These results strongly support the hypothesis that elongation in the major direction occurs directly from disks as the protein source. It is difficult to seewhy addition of single subunits, each binding to only three nucleotides, should produce regular bands with a dominant period of approximately 100 nucleotides, while this is exactly what was predicted for the addition of disks. Since the elongation is stopped abruptly while it is in full progress, the finding of 50 as well as lOO-nucleotide steps is not, surprising, it corresponds to the incorporation of only the first ring of a disk before the nuclease acted. However, we do not yet know what factor controls the relative dominance of either the 50 or the lOO-nucleotide steps and the relative weight of the bands varies between experiments. Both this point and the effect of adding other protein aggregates, such as extra A-protein, are currently under investigation. We Medical
thank Dr Research
F. E. Barralle for the Council for a research
Medical Research Council Laboratory Hills Road, Cambridge CB2 2&H, Received
24 July
1978
generous studentship
of Molecular England.
gift
of rabbit globin mRNA, to one of us (G.P.L.).
Biology
and
the
J. G. BUTLER G. P. LOMONOSSOFF
P.
LETTERS G
TO
THE
EDITOR
881
ab
3ooc Cl. c2 RI c3
2750
1960 R2
1260
c4
GI G2 15
30 MobWty
km)
FIG. 2. Size determmation for the protected RNA during TMV reassembly. The particles produced after reassembly for 1 min were digested with micrococcal nuoleartb for 5 min (track a) or 60 min (track b). Size markers are oowpea ohlorotic mottle virus RNA (track C = 3660, 3095, 2630 and 990 nuoleotides), E. coli ribosomal RNA (track R =z 3100 and 1580 nuoleotides) and rabbit globin mRNA (track G = 650 and 610 nuoleotides, allowing for oligo(A) tails of about 60 residues). The position of the main TMV RNA bands, together with sornc’ of their sizes are marked to the right of the photograph of the gel, broken bars indicating the positions of weak bands. The size calibration curve is shown on the right, with the positions of the marker RNAs shown as circles and the TMV RNA bands a~ vertical lines corresponding t.0 t,heir mobilities in the agarose/acrylamide gel. The straight line on this logarithmic-linear plot was fitted by least-squares regression analysis and the standard deviation of the slope was *4x. Thts points corresponding to RNA2 of oowpea ohlorotio mottle virus (3096 nuoleotides) and the longer ribosomal RNA (3100 nuoleotides) cannot be distinguished as their mobilities in the gel are thts same.
REFERENCES Bancroft, J. 13. (1971). Virology, 45, 830-834. Branlant, C., Sri Widada, J., Krol, A. & Ebel, J. P. (1976). Nucl. Acids Res. 3, 1671-1687. Butler, P. J. G. (1972). J. Mol. Biol. 72, 25-35. Butler, P. J. G. & Durham, A. C. H. (1977). Achan. Protein Chem. 31, 187-251. Butler, P. J. G. & Finch, J. T. (1973). J. Mol. Biol. 78, 637-649. Butler, P. J. G. & Klug, A. (1971). Nature New Biol. 229, 47-50. Butler, P. J. G., Bloomer, A. C., Bricogne, G., Champness, J. N., Graham, J., Guilley, H., Klug, A. & Zimmern, D. (1976). In Structure-l%m&on Relationship of Proteins (Markham, R. & Horne, R. W., eda), 3rd John Innes Symposium, pp. 101-110. North Holland-Elsevier, Amsterdam. Butler, P. J. G., Finch, J. T. & Zimmern, D. (1977). Nature (London), 265, 217-219. (:hampness, J. N., Bloomer, A. C., Bricogne, G., Butler, P. J. G. & Klug, A. (1976). Na.ture (London), 259, 2&24.
882
P.
J.
G.
BUTLER
AND
G.
P.
LOMONOSSOFF
Durham, A. C. H. (1972). J. Mol. Biol. 67, 289-305. Durham, A. C. H. & Klug, A. (1971). Nature New Biol. 229, 42-46. Durham, A. C. H., Finch, J. T. & Klug, A. (1971). Nature New BioE. 229, 37-42. Ehresman, C., Stiegler, P., Mackie, G. A., Zimmerman, R. A., Ebel, J. P. & Fellner, P. (1975). NucZ. Acids Res. 2, 265-278. Fukuda, M., Ohno, T., Okada, W., Otsuki. Y. & Takebe, I. (1978). Proc. Nut. Acad. Sci., U.S.A. 75, 1727-1730. Lebeurier, G., Nicolaieff, A. & Richards, K. E. (1977). Z’roc. Nut. Acad. Sk., U.S.A. 74, 1499153. Lomonossoff, G. P. 8.x Butler, P. J. G. (1979). Eur. J. Biochem. In the press. Peacock, A. C. & Dingman, C. W. (1968). Biochemistry, ‘7, 668-674. Proudfoot, N. J., Gillam, S., Smith, M. & Longley, J. I. (1977). Cell, 11, 807-818. Zimmern, D. (1975). NucZ. Acids Res. 2, 1189-1201. Zimmern, D. (1977). Cell, 11, 463-482. Zimmern, D. & Butler, P. J. G. (1977). Cell, 11, 455-462. Zimmern, D. & Wilson, T. M. A. (1976). FEBS Letters, 71, 294-298.
Quantized Incorporation of RNA During Assembly of Tobacco Mosaic Virus from Protein Disks Reassembly of tobacco mosaic virus from the isolated RNA and protein, supplied as a disk preparation consisting of over 75% as the disk aggregate, has been followed by the protection of the RNA from nuclease digestion. The sizes of the RNA fragments were determined on agarose/acrylamide gels. During the f&t few minutes the protected RNA is found to be “quantized” into discrete lengths, differing on average by about 50 or 100 nucleotides, corresponding to one or two turns of the virus helix and strongly supporting the llypothesis that elongation in the major direction, towards the 5’-hydroxyl end, is occurring by the direct addition of protein disks. Protected RNA of the full length found in tobacco mosaic virus is visible within six minutes of starting reassembly, and by 30 minutes most of the RNA is fully protected.
Under conditions where assembly of tobacco mosaic virus occurs readily and rapidly from it,s isolated RNA and coat protein, the protein exists as a mixture of small aggregates, called A-protein, and larger disk aggregates. These disks consist of two rings. each of 17 protein subunits (Durham et al., 1971; Durham & Klug, 1971), and are essential for the nucleation of virus assembly (Butler & Klug, 1971). Moreover, it appeared from the kinetics of the growth that they might be acting directly as the protein source during the rod elongation, as well as during the nucleation (Butler. 1972). This latter hypothesis has, however, been the subject of considerable debate (for a review see Butler & Durham, 1977). A major cause of this controversy was the apparent topological complexity in adding a closed ring or disk onto the end of a rod from which the free RNA tail would be protruding. This had been recognized from the start but the preliminary data indicated that disks were used during the elongation (Butler & Klug, 1971). More recently, from the combination of our studies on the nucleation region of TMVTRNA (Zimmern & Wilson, 1976; Zimmern & Butler, 1977 ; Zimmern, 1977) and on the structure of the disk aggregate (Champness et al., 1976), we were able to propose a hypothesis for t’he nucleation which largely overcomes this difficulty (Butler et al.. 1976). This involves the insertion of a special hairpin loop of the RNA into the central hole of the disk, from where it can bind round into the open “jaws” between the two rings of protein. This interaction would cause the disk to dislocate into a short helix, with the RNA entrapped between the turns of protein. This mode of nucleation results in a growing helix with both RNA tails protruding from one end, one directly and the other after doubling back down the central hole of the rod. We have confirmed that the major tail (the 5’-hydroxyl) is doubled back and that this structure is important for rapid elongation of the particles (Butler et al., 1977), and the two tails at the one end have also been pictured on partially assembled rods (Lebeurier et aE., 1977). Given this structure, elongation can, like the nucleation, occur by insertion of a loop of RNA into the jaws of a disk from its central hole; only now instead of the t Abbreviation
used:
TMV,
tobacco
mosaic
virus. 877
1,022-~X:j6/7x/:~6OX77--06
$02.00/O
:C) 1978 Academic
Press
Inc.
(London)
Ltd.
878
P.
J.
G.
BUTLER
AND
G.
P.
LOMONOSSOFF
special nucleation loop it is a “travelling loop” formed as the RNA turns back down the central hole of the rod. This loop is self-perpetuating as it can be reformed as the RNA is used up, by pulling more of the 5’-tail up the central hole. Such a mechanism of elongation makes it simple to comprehend how a complete disk can add during the elongation but it is, however, still argued that this is too complex a process to occur (Fukuda et al., 1978). We now present evidence from a direct method to show the involvement of disks during elongation. If subunits are added directly from a disk, one would expect the RNA to be incorporated in lengths of about 50 nucleotides (3 nucleotides per subunit x 17 subunits) or 100 nucleotides at a time, as one or both rings interact. During the isolation of the nucleation region of TMV RNA, which was undertaken by reacting the RNA with small amounts of a disk preparation and subsequently removing the unprotected tails with nuclease (Zimmern & Butler, 1977), a rough banding pattern suggestive of such a step-wise addition had indeed been seen (Zimmern, 1977). We have non investigated this banding in more detail over a considerably greater length of the RNA, to ensure that it does not occur only during the init,ial steps which were already known to require disks. The measurement of the lengths of the protected RNA is used as a direct measure of the elongation. Reassembly was carried out at 20°C in sodium phosphate buffer (pH 7-O), ionic strength 0.1 M> with a protein disk preparation at 8 mg/ml and RNA at 0.2 mg/ml final concentration. Preparations were as previously described for both the protein (Durham, 1972) and the RNA (Zimmern, 1975). Elongation was stopped and the uncoated RNA removed by the addition of CaCl, and micrococcal nuclease to 1 mM and 40 units/ml, respect’ively. This concentration of Ca2+ alone was found to totally inhibit elongation in some preliminary experiments. After digestion at 20°C for the desired time, 1,2-di(2-aminoethoxy)-ethane-N,N,N’,Nf,-tetraacetic acid was added to 5 mM to complex the Ca2 + , and the rodlets pelleted at 55,000 revs/min and 5°C for five hours in an M.S.E. 10 x 10 ml titanium rotor. The rodlets were resuspended in lOOmMNaCl, 1 mM-EDTA, lOmr\l-Tris-chloride (pH7.5), and the RNA extracted as before and precipitated twice with ethanol to remove the phenol. RNA lengths were determined by electrophoresis in 0.5% agarose/2.5% polyacrylamide gels prepared and run according to Peacock & Dingman (1968), but) with 0.1% sodium dodecyl sulphate added to all buffers. Samples were denatured just’ prior to application to the gel by heating to 100°C in 50% (v/v) formamide containing 10 mM-EDTA. After electrophoresis, the gels were soaked for 30 minutes in water, stained with 2 mg ethidium bromide/l and briefly washed in water again before photographing under short wavelengt,h ultraviolet light through a red filter. A time course for the elongation is shown in Figure 1, and shows a clear banding pattern and the steady elongation. Full length RNA is first protected by six minutes, in agreement’ with our previous estimates of the maximum rate of growth (Butler, 1972; Butler & Finch, 1973), and within 30 minutes the strong band is full length and the amount of smaller material is very similar to that in the original TMV RNA sample, showing that the reaction is complete within this time. This is in contrast to t,he longer times apparently required with OM-strain RNA and protein (Fukuda et al., 1978), and suggests that results from this strain should not be compared directly with those from the commonly used Vulgare strain. The lengths were estimated for the bands by extrapolation from those of the Escherichia coli ribosomal RNAs, which were taken as 1580 nucleotides (Ehresman et al., 1975) and 3100 nucleotides (Branlant
LETTERS R
I
TO
THE 4
2
3
COUI‘BR
of elongation
EDITOR 6
X7!l IO
15
30
T
R
6400
1230
470
FIG.
1. Time
of protected
RNA.
out for the times indicated (in min) and the uncoated tails remowd I,> Reaf ISA mbly was carried micrococcal nuclease for 30 min. E. coli ribosomal RNA was used as a markcI dig&it “ll with (track TMV RNA preparation used for the elongation was also examinrvl RI and the original banding pattern, together with some approximate corrected lengths (SW (track T) . The regular is indicated on the left., broken bars indicating t,he positions of weak bands. tlisscus iSi In in text),
et al.: 1976), plotting the logarithm of the length against the mobility. Because of the long extrapolation and reliance on only two markers, these sizes will not be accurattb but, t,o a first approximation, they do showa spacing of approximately 50 nucleotides over the whole range from 300 to 1000. Later experiments with many more markers (Fig. 2) show that the shorter ribosomal RNA migrates rather slower than expected for its size. If allowance is made for this, the spacing becomes about 54 nucleotides over the range from 470 to 1230. The corrected sizes are shown in Figure 1, and this is justified by the obvious similarities between the band patterns in the separate experiments shown in Figures 1 and 2. One noticeable feature of the RNA lengths is the wide range of sizes present at an?time. Taken together with the build-up of certain bands, this suggests that secondary
880
P.
J.
G.
BUTLER
AND
G.
P.
LOMONOSSOFF
structure of the RNA may be delaying the elongation at these points, most probably by obstructing the pulling of the RNA tail up the central hole of the rod. A further feature is the loss of discrete bands by about ten minutes. This must be the result of a spreading of the lengths of rods present and is presumably due to the loss or accretion of single protein subunits to or from the A-protein present in the disk preparation, rather than a strict addition only of intact disks to the growing particles. The most likely place for this to occur is at the minor 3’-tail, which is not doubled back down the rod but protrudes directly. Despite the reports that the 3’-tail is not coated at all before completion of coating of the 5’-tail (Lebeurier et al., 1977 ; Fukuda et al., 1978), coating of this tail appears to be independent of the major Y-tail and to occur throughout elongation (Lomonossoff & Butler, 1979), albeit at a much slower rate than the major tail, and it may very well occur from A-protein rather than from disks, and so be unquantized and smear the pattern from the other end. In another experiment (Fig. Z), the lengths of the RNA fragments were moro accurately determined, by including markers of cowpea chlorotic mottle virus RNA (Bancroft, 1971) and rabbit globin messengerRNA (Proudfoot et al., 1977), and measuring the bands which fell within the range of the markers rather than extrapolating. Over the range from 1260to 2750 nucleotides the bands are mainly separated by steps of about 100 nucleotides (or sometimes200), with a few st,epsof about 50. Taking a weighted average over this range (to estimate the fundamental spacing) gives a step length of 99 nucleotides, with a standard deviation of &4%, in close agreement with the value of 102 nucleotides predicted for the length covered by the addition of complete disks. It is unlikely that the banding pattern is due to an artefact of the nucleasedigestion, such as unfavourable sequences.Micrococcal nuclease will cleave either single 01 double-stranded nucleic acids and is not very specific in sequencerequirement. The regular pattern of the band shows that such sequences,if they exist, would have to be repeated over a length of more than 1500 nucleotides, or roughly a quarter of the viral RNA. Moreover, no significant difference is visible in the patterns produced after 5 or 60 minutes digestion (Fig. 2, tracks a and b) showing that the pattern is not simply a result of underdigestion. These results strongly support the hypothesis that elongation in the major direction occurs directly from disks as the protein source. It is difficult to seewhy addition of single subunits, each binding to only three nucleotides, should produce regular bands with a dominant period of approximately 100 nucleotides, while this is exactly what was predicted for the addition of disks. Since the elongation is stopped abruptly while it is in full progress, the finding of 50 as well as lOO-nucleotide steps is not, surprising, it corresponds to the incorporation of only the first ring of a disk before the nuclease acted. However, we do not yet know what factor controls the relative dominance of either the 50 or the lOO-nucleotide steps and the relative weight of the bands varies between experiments. Both this point and the effect of adding other protein aggregates, such as extra A-protein, are currently under investigation. We Medical
thank Dr Research
F. E. Barralle for the Council for a research
Medical Research Council Laboratory Hills Road, Cambridge CB2 2&H, Received
24 July
1978
generous studentship
of Molecular England.
gift
of rabbit globin mRNA, to one of us (G.P.L.).
Biology
and
the
J. G. BUTLER G. P. LOMONOSSOFF
P.
LETTERS G
TO
THE
EDITOR
881
ab
3ooc Cl. c2 RI c3
2750
1960 R2
1260
c4
GI G2 15
30 MobWty
km)
FIG. 2. Size determmation for the protected RNA during TMV reassembly. The particles produced after reassembly for 1 min were digested with micrococcal nuoleartb for 5 min (track a) or 60 min (track b). Size markers are oowpea ohlorotic mottle virus RNA (track C = 3660, 3095, 2630 and 990 nuoleotides), E. coli ribosomal RNA (track R =z 3100 and 1580 nuoleotides) and rabbit globin mRNA (track G = 650 and 610 nuoleotides, allowing for oligo(A) tails of about 60 residues). The position of the main TMV RNA bands, together with sornc’ of their sizes are marked to the right of the photograph of the gel, broken bars indicating the positions of weak bands. The size calibration curve is shown on the right, with the positions of the marker RNAs shown as circles and the TMV RNA bands a~ vertical lines corresponding t.0 t,heir mobilities in the agarose/acrylamide gel. The straight line on this logarithmic-linear plot was fitted by least-squares regression analysis and the standard deviation of the slope was *4x. Thts points corresponding to RNA2 of oowpea ohlorotio mottle virus (3096 nuoleotides) and the longer ribosomal RNA (3100 nuoleotides) cannot be distinguished as their mobilities in the gel are thts same.
REFERENCES Bancroft, J. 13. (1971). Virology, 45, 830-834. Branlant, C., Sri Widada, J., Krol, A. & Ebel, J. P. (1976). Nucl. Acids Res. 3, 1671-1687. Butler, P. J. G. (1972). J. Mol. Biol. 72, 25-35. Butler, P. J. G. & Durham, A. C. H. (1977). Achan. Protein Chem. 31, 187-251. Butler, P. J. G. & Finch, J. T. (1973). J. Mol. Biol. 78, 637-649. Butler, P. J. G. & Klug, A. (1971). Nature New Biol. 229, 47-50. Butler, P. J. G., Bloomer, A. C., Bricogne, G., Champness, J. N., Graham, J., Guilley, H., Klug, A. & Zimmern, D. (1976). In Structure-l%m&on Relationship of Proteins (Markham, R. & Horne, R. W., eda), 3rd John Innes Symposium, pp. 101-110. North Holland-Elsevier, Amsterdam. Butler, P. J. G., Finch, J. T. & Zimmern, D. (1977). Nature (London), 265, 217-219. (:hampness, J. N., Bloomer, A. C., Bricogne, G., Butler, P. J. G. & Klug, A. (1976). Na.ture (London), 259, 2&24.
882
P.
J.
G.
BUTLER
AND
G.
P.
LOMONOSSOFF
Durham, A. C. H. (1972). J. Mol. Biol. 67, 289-305. Durham, A. C. H. & Klug, A. (1971). Nature New Biol. 229, 42-46. Durham, A. C. H., Finch, J. T. & Klug, A. (1971). Nature New BioE. 229, 37-42. Ehresman, C., Stiegler, P., Mackie, G. A., Zimmerman, R. A., Ebel, J. P. & Fellner, P. (1975). NucZ. Acids Res. 2, 265-278. Fukuda, M., Ohno, T., Okada, W., Otsuki. Y. & Takebe, I. (1978). Proc. Nut. Acad. Sci., U.S.A. 75, 1727-1730. Lebeurier, G., Nicolaieff, A. & Richards, K. E. (1977). Z’roc. Nut. Acad. Sk., U.S.A. 74, 1499153. Lomonossoff, G. P. 8.x Butler, P. J. G. (1979). Eur. J. Biochem. In the press. Peacock, A. C. & Dingman, C. W. (1968). Biochemistry, ‘7, 668-674. Proudfoot, N. J., Gillam, S., Smith, M. & Longley, J. I. (1977). Cell, 11, 807-818. Zimmern, D. (1975). NucZ. Acids Res. 2, 1189-1201. Zimmern, D. (1977). Cell, 11, 463-482. Zimmern, D. & Butler, P. J. G. (1977). Cell, 11, 455-462. Zimmern, D. & Wilson, T. M. A. (1976). FEBS Letters, 71, 294-298.