Eur. J. Biochem. 54,145- 153 (1975)

Observations Concerning the Sequence of Two Additional Specifically Encapsidated RNA Fragments Originating from the Tobacco-Mosaic-Virus Coat-Protein Cistron Hubert GUILLEY, Gerard JONARD, Kenneth Eugene RICHARDS, and Leon HIRTH Laboratoire des Virus des Plantes, Institut de Biologie Moleculaire et Ccllulaire du Centre National de la Recherche Scientifique, Strasbourg (Received September 2, 1974iJanuary 27, 1975)

The incubation of 25-S tobacco mosaic virus (TMV) protein with a mixture of RNA fragments produced by partial TI RNase digestion of TMV RNA results in the encapsidation of only a few species of RNA. In addition to the most predominant species, fragment 1, whose sequence has been described in the preceding paper, two other species, fragment 41 and fragment 21, are coated by the protein. These two RNA fragments were purified by polyacrylamide gel electrophoresis and subjected to total digestion with pancreatic and T, RNase. The oligonucleotides were separated by paper electrophoresis and characterized insofar as possible by digestion with the complementary ribonuclease. From the amino acid coding capacity of the oligonucleotides liberated from fragments 41 and 21 by T, RNase digestion, it appears that these two fragments, like fragment 1, are derived from the coat protein cistron. They are situated immediately prior to fragment 1 and, together with this fragment, constitute a continuous stretch of 232 nucleotides of the cistron which codes for amino acids 53 to 130 of the coat protein. The order of the fragments in the sequenceis 21 - 41 - 1. A possible model for the secondary structure of this portion of the sequence is proposed.

Recently, we have shown that the 25-S tobacco mosaic virus (TMV) protein aggregate can combine rapidly and quantitatively with several TMV RNA fragments obtained by partial TI RNase digestion of TMV RNA [l]. The nucleotide sequence of one of these fragments, fragment 1, has already been established [2,31and has led us to deduce that it corresponds to a portion of the TMV coat protein cistron [4]. This particular fragment is referred to as “specifically encapsidated RNA fragment A”. In the course of the sequence work on this fragment, small quantities of two additional specifically encapsidated fragments were isolated and purified by polyacrylamide gel electrophoresis. The were each subjected to total TI and pancreatic RNase hydrolysis and the digestion products were partially sequenced. Analysis of the amino acid coding capacity of the digestion products has revealed that the two parent fragments (fragments 41 and 21) originate from the portion of the coat protein cistron immediately adjacent to fragAhhreviution. TMV, tobacco mosaic virus. Enzymes. Pancreatic ribonuclease (EC 3.1.4.22); T, ribonuclease

(EC 3.1.4.8). Eur. J. Biochem. 54 (1975)

ment 1. Fragment 21 codes for amino acids 53 to 76 and fragment 41 for amino acids 77 to 94 of the coat protein. With the aid of the known sequence of the coat protein it has proved possible to order the products of total enzymic digestion so as to allow the complete sequences of the two fragments to be deduced.

MATERIALS AND METHODS Preparation of 32P-labeledTMV RNA and TMV protein, T, RNase partial hydrolysis of TMV RNA, reconstitution and isolation of the specifically encapsidated RNA fragments were done as previously described [2,3]. After purification by polyacrylamide gel electrophoresis the RNA fragments were hydrolyzed to completion with either T, or pancreatic ribonuclease. The conditions of hydrolysis have been described in the preceding paper [3]. The oligonucleotides were characterized by the methods developed by Sanger and coworkers [5,6].

Sequence from the TMV Coat-Protein Cistron

146

RESULTS

A

6

Isolation, Purificution and Sequence Analysis c?f'Frugment41 and 21

We have shown elsewhere [2,3] that only a few of the RNA fragments which are liberated by limited T1 RNase digestion of TMV RNA are encapsidated by the TMV protein disk. After elimination of the RNA molecules which are not recognized, the specifically encapsidated fragments may be separated from one another by electrophoresis upon a 10% poly acrylamide gel (Fig. 1A). The nucleotide sequence of fragment 1 (Fig. 1A) has been described in the preceding paper [3]. This fragment will also be referred to as "specifically encapsidated RNA fragment A". Fragments 3, 5 a and 5 b were shown to be derived from the sequence of fragment 1 by an additional scission in the nuclcotide chain [3]. The material contained in bands 2 and 4 was not homogeneous and in each case several components could be resolved by electrophoresis upon 15 yd) gels (Fig. 1 B). Analysis of the oligonucleotide composition of the purified bands revealed that fragment 22 is identical to fragment 3 except for the presence of an additional A-U-G at its 5' extremity and that fragments 42 and 43 correspond to the two halves of fragment 1. Fragments 41 and 21 were homogeneous after electrophoresis upon thc 15% gel. Portions of the purified fragments were subjected to total hydrolysis with paiicrcatic and with TI ribonuclease and the products of' digestion in each case were separated by two-dimensional electrophoresis according to Sanger et al. [5].The fingerprints are shown in Fig. 2A and 2 b (fragment 41) and Fig. 2c and 2d (fragment 21). Each of the oligonucleotides released from the two RNA fragments by total digestion with one or the other cnzyine was then digested with the complementary nuclease and the final products were characterized by electrophoresis at pH 1.9 [3]. From this information the sequence of each original oligonucleotide was constructed insofar as possible. Table 1 lists all the oligonucleotides found in the total T, and pancreatic RNase digests of fragments 41 and 21 and the molarity of each oligonucleotide in its parent fragment. It is apparent that in overall oligonucleotide composition fragmcnts 41 and 21 differ markedly from one another and from fragment 1. The three fragments must therefore originate from different parts of the RNA molecule. Not enough radioactivity was present in the purified fragments 41 and 21 to allow a complete sequence analysis by the examination of products of partial digestion. Small portions of each sequence can be reconstructcd, howevcr, by consideration of the total digestion products. The oligonucleotides P9 and T8

0 1

rH

21 22

I

2

3 4

15% GEL

I

5 a,b

41 42 43

6

+-

Br

Fig. 1. Polyairj~lurnidegel clr~crroplmrcsis(!/ nparrial 7', ribonuclense diges/ of TMT' RNA &r rcwna/i/u/ion 14 ith 25-5' TM I' jirorrirz. The bands have been v i s u d h d by autoradiopraphy. Digesiion of TMV [32P]RNA was done with an ctizyitic: KNA ratio 1 : 150 (by weight) in sodium pyrophosphate I = 0.5. pH 7.25. for 30 min at 0°C. The enzyme was eliminated by phenol extraclion and 2 . 5 3 TMV protein was added to the fragmented RNA (with a protein:RNA ratio of 1 :?). After 4 h reconstitution at 24 C, the reconstituted material was purified by ultraceiitrifugation. and the RNA was extracted and loaded onto a l00:, polyacrylarnide gel (A). 'The fractionated fragments are numbcred. Fragment 0 and 6 are not alwayspresent and their intensity is orten low. In this experiment, fragment 5 is separated into two bands (5a and 5h), the two fragments having the same sequence with the exception that 5a ~ O S S K S S K San additional A-LJ-G at the 5' end. Fragments 2 and 4 were purified on 15 " b polyacrylamide gels (B) yielding scveral new fragments which have also been nufllhered. The location of the specifically cncapsidated RNA fragments 1, 41 and 21 are indicated in the figure. Br indicates the position of hroniophcnol hluc

from fragment 41, for instance, must be joined endto-end in the parent fragment to yicld the sequence Y-A-G-A-A-U-A-A-U-A-G. In the same manner, oligonucleotides P10 and T7 from fragment 41 could be arranged to give the sequence Y-A-G-A-A-AU-A-G. Origin of Fragments 41 and 21

Fragment 1 is known to correspond to a portion of the coat protein cistron, that which codes for amino Eur. J. Btochem. 54 (1975)

H. Guilley, G. Jonard, K. E. Richards, and L. Hirth

Fig.2. Fingerpinis of tho RiVuse digcsrs of fiugnwnrs 41 and 21. (a) Pancreatic RNase digest of fragment 41 : (b) TI RNase digest of fragment 41 ; (c) pancreatic RNase digest of fragment 21 ; (d) TI RNase digest of fragment 21. The conditions of hydrolysis have been described in the preceding paper [3]. Products PI2 from fragment 41 and P15 from fragment 21 were present upon the fingerEur. J. Biochcm. 54 (1975)

147

prints as three separate spots, the intensities of which are too low to be detected in the reproduction. Analysis of the separated products established that they were identical in sequence. the sequence being A-G in each case. Electrophoresis was done on cellulose acetate pH 3.5 (arrow 1) and on DEAE-cellulose paper in 77” formic acid (arrow 2)

Sequence from the TMV Coat-Protein Cistron

148

Table 1. T, mid pancreatic ribonuclease digestion products from fragments 31 and 21 The relative molar yield of each oligonucleotide is also given. The complete sequence of some oligonucleotides was deduced from the information given by the complementary enzymc digestion products and, in the case of oligonucleotides marked with an asterisk (*), from the amino acid sequence of the portion of the coat protein for which it codes. exp. = experiment yield. th. = theoretical yield Fragment 41

T, ribonuclease products

pancreatic ribonuclease products spot

scquencc

cxp.

th.

spot

sequ encc

molichain P1 P2 P3 P4 P5 P6 P7 PE; P9 PI0 PI1 P12

CJ C

4-c

G-C G-A-C + A-G-C A-U A-A-U A-G-U .A-G-A-A-U A-G-A-A-A-U ,4-G-G-U* A-G

6.7 2.6 3.8 2.0 2.1 2.2 1.4 1.3 1.0 1.0 2.0 0.8

exp.

th.

mol/chain

6 3 4 2 2 2 1

1 1 1 1 1

T1 T2 T3 T4 TS T6 TI T8 T9 T10

G A-C-C-C-G* U-G C-U-A-G* U-C-A-C-A-G* A-C-A-C-U-A-Ci* A-A-A-U-A-G A-A-U-A-A-U-A-G C-A-U-U-U-G* C-A-U-U-A-C-U-A-G*

0.8 0.9 1.o 1.0 0.9 0.9 1.0 1.0 0.9 0.8

1 1 1 1 1 1 1 1 1 1

exp.

th.

Fragment 21

T, ribonuclease products

pancreatic ribcmuclease products spot

iequence

exp .

th.

spot

sequence

mol/chain P1 P2 P3 P4 P5 P6 PI P8 P9

PI0 P11 P12 PI 3 I’14 P15

t.1

c

.A-C

A-A-C A-A-A-C G-C (;-A-C A-A-U

c;-u

A-G-U A-A-G-tJ GG-U A-G-G-U* ,4.A-(3-(3-U* A-G

moljchain

9.0 4.8 4.2 0.9 1.1 1.0 1.9 1 .0 2.9

10 6 4 1 1 1 2 1 3

T1 T2 T3 T4 T5

1.1 1 .o 0.9 2.0

1

TI0 TI 1

1.1

0.7

1 1 2 1 1

acids 95 - 130 of the coat protein [4]. It seems conceivable that the other two specifically encapsidated fragments, 41 and 21, might also derive from the coat protein cistron. This hypothesis can be tested, even without knowledge of the complete sequences of these fragments, by examination of the amino acid coding capacity of their parts, that is the TI RNase digestion products of the two fragments. An example of such an analysis for the sequence . . . Y-A-G-A-AU-A-A-U-A-G . . . from fragment 41 (see above) is shown in Fig. 3 A. In one of the three possible reading

T6 T7 T8 T9

G C-G A-C-A-G

3.6 1.2 0.9

U-G

2.8 0.9 2.0 0.9 0.9 0.6

U-A-C-A-G* U-U-A-G U -A-A-C-U -G* U-A-C-A-A-U-G* A-A-A-C-C-U-U-C-AC-C-A-C-A-A-G* U-U-C-C-C-U-G* A-C-U-U-U-A-A-G*

0.7 0.7

4 1 1 3 1

2 1 1 1 1

z

frames (frame 2) this stretch of bases could be translated as . . . Arg-Ile-Ile . . . which corresponds to the amino acid sequence at positions 92-94 of the coat protein [7]. The peptide sequences resulting from translation in either of the other two frames are not present in the coat protein. In many instances the sequence of the T I RNase oligonucleotides was not known with certainty and it was necessary to consider the amino acid coding capacity of all allowable permutations of the sequence. Fig. 3 B shows such an analysis for oligonucleotide bur. 1. Biochcm. 54 (1975)

€1. Guilley, G. Jonard, K. E. Richards, and L. Hirth

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3

A

t_-

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149

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3.

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