Eur. J. Biochem. 54, 135- 144 (1975)

Sequence of a Specifically Encapsidated RNA Fragment Originating from the Tobacco-Mosaic-Virus Coat-Protein Cistron Hubert GUILLEY, Gtrdrd JONARD, Kenneth Eugene RTCHARDS, and LCon HIRTH Laboratoire des Virus des Planks, Institut de Biologie Moltculaire ct Cellulaire du Centre National de la Recherche Scientifique, Strasbourg (Received September 2, 1974/January 27, 1975)

When 25-S tobacco mosaic virus (TMV) protein aggregate and TMV RNA, which has been partially digested by TI RNase, are mixed under conditions suitable for reconstitution, only a few RNA fragments are encapsidated. These fragments were isolated and purified by polyacrylamide gel electrophoresis. The sequence of the three main fragments, the longest of which (fragment 1) was estimated to contain 103 nucleotides, has been determined. The two smaller fragments are portions of the longer chain produced by an additional specific scission. Because of the great affinity of 25-S TMV protein for this nucleotide sequence, it will be referred to as the “specifically encapsidated RNA fragment-’. The occurrence of a “hidden break” in the sequence has been demonstrated: fragment 1, purified by electrophoresis on a polyacrylamide gel without 8 M urea, gives rise upon further electrophoresis in the presence of urea to two new bands corresponding to the two halves of the molecule. A stable hair-pin secondary structure has been derived from the base sequence which can account for the specificity of action of the enzyme. Because of its properties, we have suggested elsewhere that the sequence of fragment 1 might correspond to the disk recognition site for reconstitution, which is known to be located at the 5‘ end of the intact RNA. But experiments with TMV RNA whose 5‘-OH end has been radioactively phosphorylated with polynucleotide kindse show that this is not the case. Analysis of the amino acid coding capacity of the fragment has instead revealed that fragment 1 is a portion of the TMV coat protein cistron.

The reconstitution in v i m of tobacco mosaic virus (TMV) from its RNA and protein components is a polar process, starting at or near the 5’-OH end of the RNA chain [1,2]. The initial step in assembly involves an interaction between a double disk of TMV protein and the 5’-terminal region of the RNA chain 131. Reconstitution, under normal conditions, is quite specific for TMV RNA [4,5]. It is generally believed that this specificity arises at the initiation stage of the reaction, i.e., that the double disk has a great affinity for some unique feature, either of sequence or secondary structure, at the 5’-terminus of the TMV RNA chain. The “recognition site” must, furthermore, be rather limited Abbreviation. TMV, tobacco mosaic virus ATP : Adenosine triphosphate. Enzymes. Pancreatic ribonuclease (EC 3.1.4.22);T, ribonuclease (EC 3.1.4.8); bacterial alkaline phosphatase (EC 3.1.3.1): snake venom phosphodiesterase (EC 3.1.4.1); polynucleotide kinase (EC 3.1.3.33).

Eur. J. Biochem. 54 (1975)

in extent since removal of 10 [6] to 50 nucleotides [3] from the 5’-terminus effectively prevents initiation. Determination of the sequence of the double disk recognition site is essential for a full understanding of the properties which govern its special affinity for the disk. In a previous publication we have briefly reported the isolation and sequence of a 103-nucleotide fragment of TMV RNA which combines rapidly and irreversibly with the double disk [7]. This paper describes in more detail the sequencing and some of the properties of this specifically encapsidated RNA fragment (fragment 1). In view of its great affinity for the double disk, we have suggested that it might correspond to the disk recognition site located at the 5’ terminus of the RNA chain. In this paper, experiments designed to test this hypothesis, using RNA whose 5‘ end had been marked with radioactive phosphate, will be described.

136

Sequence of a Specifically tncapsidated TMV RNA Fragment

MATERIALS A N D METHODS

Polyucrylamide Gel Electrophoresis

Preparation of 32P-LabeledT M V RNA

The RNA fragments obtained by partial T, RNase hydrolysis of TMV RNA were separated on 1OYo polyacrylamide gel slabs [ l l ] for about 16 h at 4° C in the presence of electrophoretic buffer containing 0.1 M Tris-boratc pH 8.3, 2.5 mM EDTA and 8 M urea. Partial TI RNase digests of purified RNA fragments were fractionated on 15 to 24'x polyacrylamide gels. Fractionated fragments were detected by autoradiography. On the basis of the autoradiogram the fragments were excised and eluted electrophoretically onto discs of DEAE-cellulose paper as described by Adams et al. [I 11. The fragments were then eluted from the paper with triethylamine bicarbonate (which must be at pH 10).

Nicotiana tubaccum var. Judy Pride infected for two days with TMV were removed from their pots; their roots were dipped in deionized water containing 1 mCi of "P-labeled phosphoric acid per ml. The leaves were harvested after six days and the virus was extracted and purified by poly(ethy1ene glycol) precipitation [XI. Extraction of 32P-labeled RNA from TMV was performed by the phenol method [9]. The RNA generally possessed a specific radioactivity of 0.05-0.075 pCijpg. This specific activity is 3050 times less than that of the bacteriophage RNAs or ribosomal RNAs which have been the subject of sequence analysis by others. Consequently it has been difficult and sometimes impossible to establish with absolute certainty, the sequence of long oligonucleotides. Preparation of T M V Protein TMV protein was prepared by the acetic acid procedure [lo]. Before use in reconstitution experiments the 4-S TMV protein was transformed into 25-S TMV protein by addition of sodium pyrophosphate, pH 7.25. to yield a final ionic strength of 0.5.

Partiul 7; Ribonuclease Hydrolysis of TMV R N A TMV RNA in the reconstitution buffer (i.e. sodium pyrophosphate, pH 7.25, I : 0.5 M) was hydrolyzed with 1 unit of T, ribonuclease per 50 pg of RNA for 30 min at 0 'C. The enzyme was extracted with an equal volume of redistilled phenol saturated with water. The digest was then used in reconstitution experiments or charged directly onto 10 polyacrylamide gels as described below.

Partiul Ribonuclease Hj~drolysis of RNA Fragments Isolated by Gel Electrophoresis For partial hydrolysis with TI ribonuclease, the RNA fragments mixed with 50 pg carrier yeast RNA, were digested with 0.3 unit of T, ribonuclease in 30 p1 sodium pyrophosphate buffer pH 7.25, I : 0.5 M at 0°C: for 10 min. Digestions were terminated by beginning gel electrophoresis of the products immediately. For partial hydrolysis with pancreatic ribonuclease, the oligonucleotides were digested for 10 min at 0°C with an enzyme: substrate ratio of 1 : 500 in 20 pl of 0.01 M Tris-HC1, pH 7.4, 0.001 M EDTA and then spotted on DEAF,-cellulose paper. The products were separated by electrophoresis in 7 % (v/v>formic acid.

Complete TI and Pancreatic Ribonuclease Hjdruolysis Complete T, or pancreatic RNase digestions of the RNA fragments extracted from the gels were performed in 10 p1 of 0.01 M Tris-HC1 pH 7.4, 0.001 M EDTA containing 50 pg of ycast RNA and either 0.25 pg pancreatic RNase or 2 units TI RNase. Incubation was for 30 min at 37 "C. The digests were separated on strips of cellulose acetate in 5 % (viv) acetic acid, 0.5 :( pyridine pH 3.5 and transferred onto Whatman DE-81 paper [12,13]. Separation of the products in the second dimension was achieved by electrophoresis in 7 (v/v) formic acid.

Sequence Analysis of Oligonucleotides Oligonucleotides were recovered from the fingerprints by the method of Sanger et al. [12]. For oligonucleotides originating from TI RNase, the next step in sequence determination was total pancreatic RNase hydrolysis ; for oligonucleotides originating from pancreatic RNase digestion, the next step was total hydrolysis with T, RNase. The resulting products were fractionated by electrophoresis on DE-8 1 paper at pH 1.9 (2.5 % formic acid/8.7 yo acetic acid). If the sequence was not yet solved by this stage, the oligonucleotides were hydrolyzed either with U 2 RNase or dephosphorylated with bacterial alkaline phosphatase and completely hydrolyzed with snake venom phosphodiesterase. The conditions for enzymatic digestion of oligonucleotides and separation of the products are summarized in Table 1 . Additional analysis (partial digestions of oligonucleotides with either venom phosphodiesterase or spleen phosphodiesterase) was generally impossible because of the relatively low specific radioactivity of the starting RNA. Eur. J . Biochem. 54 (3975)

137

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

Table 1. Conditionsfor enzymic hydrolysis of oligonucleotides Further digestions were performed by addition of 10 pl of the appropriate enzyme solution (described below) to the RNA fragments. 50 pg of tRNA was generally added lo serve as a carrier. Incubation was performed at 37°C. Electrophoresis was done on Whatman DE-81 paper, except for venom phosphodiesterase digests, which were separated on Whatman 3MM paper

Enzyme

Enzyme concn

Pancreatic ribonuclease Bacterial alkaline phosphatase Snake venom phosphodiesterase T, ribonucleasc

0.025 0.6 0.2 0.1

Buffer

Time of incubation

mg/rnl

rnin 0.01 M Tris-HC1 pH 7.5, 0.001 M EDTA 0.01 M Tris-HCI pH 7.5, 0.001 M EDTA 0.025 M Tris-HCI pH 8.9, 0.01 M MgC1, 0.01 M Tris-HC1 pH 7.5. 0.001 M EDTA

unit/pl

U, ribonuclease

1

0.05 M sodium acetate pH 4.5

Phosphorylation of the 5‘ Terminus of TMV R N A by Means of Polynucleotide Kinase Phosphorylation of the 5’ terminus of TMV RNA with polynucleotide kinase was performed essentially by the method of Fraenkel-Conrat and Fowlks [14]. In most cases, the RNA to be used was first fractionated by sucrose gradient centrifugation to remove degraded molecules. The kinase reaction was performed on 2mg of RNA in a final volume of 1 ml. The reaction mixture contained 0.07 M Tris, pH 7.6, 12 mM MgCl,, 6 mM 2-mercaptoethanol and 24 yM [y-3ZP]ATPwith a specific radioactivity of 2 - 3 mCi/ ymol. 5- 10 units of polynucleotide kinase from T4XF1-infected E.coliB were added and the mixture was incubated at 37 “C for 60 - 80 min. The reaction was stopped by addition of 0.2 ml 0.1 M EDTA and the mixture was extracted with phenol. After removal of phenol from the aqueous phase with ether, the RNA was precipitated with ethanol, redissolved in water and reprecipitated with an equal volume of 4 M NaC1. The RNA was dissolved in water, reprecipitated with ethanol and finally layered upon a 5-2074 sucrose gradient, containing 0.01 M sodium phosphate, 0.1 M NaCl pH 7.0. Centrifugation was for 14 h at 25000 rev./min (rotor SW 27). The gradient was fractionated and the quantity of RNA and 32P in each fraction was determined by spectrophotometry and by counting. The RNA peak fractions of lowest specific radioactivity were pooled and precipitated with alcohol. The polynucleotide kinase was judged to be free of contamination with RNase since kinasetreated and untreated RNA were found to yield identical profiles after sucrose gradient centrifugation or electrophoresis upon polyacrylamide gels. The extent of phosphorylation of the full length RNA calculated from its specific radioactivity varied from experiment to experiment, ranging between 0.4 to 1.O phosphate per full length RNA chain. Eur. J. Biochem. 54 (1975)

Electrophoresis pH

30 30 120 30

1.9 1.9 3.5 1.9

h

%formic acid

16

7

Mater ials TI ribonuclease was purchased from Calbiochem, pancreatic ribonuclease, bacterial alkaline phosphatase and snake venom phosphodiesterase from the Worthington Biochemical Corporation, and polynucleotide kinase from P.L. Biochemicals. U, RNase was a gift of Prof. J. P. Ebel. [32P]Orthophosphate (carrier-free in dilute HC1) and [ Y - ~ ~ P I Awere T P obtained from the Commissariat a I’Energie Atomique. Kodak “Rapid processing” RPjS14 X Ray films were used for autoradiography. The high-voltage electrophoresis apparatus was from Gilson Medical Electronics.

RESULTS Reconstitution of T M V 13’P]RNA Partially Digested with T I RNase A partial TI RNase hydrolysate of TMV RNA (for conditions see Materials and Methods) was mixed with 25-S TMV protein (with a protein: RNA ratio of I : 2) and incubated for 4 h at 24 “C. After ultracentrifugation (7 h at 105000 x g ) , the sedimentable material was dissolved in 0.01 M phosphate buffer pH 7.0 and centrifuged again. The pellet was resuspended in the phosphate buffer. We have shown elsewhere [15] that the purified products of reconstitution consist of two distinct size classes of short rod-like nucleoprotein particles. The RNA extracted from this material, together with a control (i.a. the total hydrolysate before reconstitution), was electrophoresed on a 10% polyacrylamide gel. A typical pattern of such a gel is shown in Fig. 1. Six main bands are present for the sample corresponding to the RNA extracted from the reconstituted material. This observation implies that only a few RNA fragments are selectively recognized by TMV protein. The same six

Sequence of a Specifically Fncapsidated 'IMV K N A bragment

A

After extraction from the gels the purified fragments were subjected to complete digestion with pancreatic and TI ribonucleases and the digests were fingerprinted. Fig. 2 shows the oligonucleotide maps of fragments 1, 3 and 5. The relative molar stoichiometry of the TI and pancreatic oligonucleotides in the three fragments were calculated from the proportion of radioactivity in each spot and the sequence of the digestion products was determined by hydrolysis with the complementary ribonuclease. The results are given in Table 2. Further sequence work on long TI oligonucleotides was carried out according to the procedure described in Materials and Methods. The results are summarized in Table 3. Some pancreatic oliganucleotide sequences were determined by overlapping of partial T, RNase hydrolysis products of fragments 3 and 5. If we compare the oligonucleotide composition of fragments 1, 3 and 5 , it appears that fragments 3 and 5 derive from fragment 1 without overlapping between them. The presence of the oligonucleotides A-G-A-U and A-U-G in the fingerprints of fragment 1 alone indicates that fragments 3 and 5 are joined together by A-G-A-U-G.

6

1

2 3 4 5

6

-

Br

p i E/WrRl[JhOWSis.Y of'upartiai T~ ribonuc/ea.re Rh'il hrfi1i-c i B I und u/iw 1.4) rrcons/ifrrrion n:ith 3 - S T M V p o r c ~ i nThe . rragments were fractionated in the presence polyacrylamide gcl containing 0.1 M Tris-

Fig.

I . PO/! Lil.i ,,/il,t?irtc.

di,ty,,s/ O / T i t

-

pH 8.3. Br indicates the position of bromophenol blue

bands are always obtained but their relative intensity varies somewhat from experiment to experiment, Frequently. band 6 is present only in minor quantities. Each band was purified on a 15 ?: polyacrylamide gel as described by Vigne and Jordan [16].

Oligomcleotide Sequences .J'TI mid Puncreutic Rihorndease Hydrolysates .f' Fvugrncvits I , 3 and 5 Electrophoresis on 15 0 4 polyacrylamide gels revealed that bands 1, 3 and sometimes band 5 were pure. Bands 2 and 4, although containing some material with sequences identical to those in bands 1, 3 and 5 , were not pure after the first electrophoresis and were not used in the sequence determinations. Band 6 contained no sequences in common with the other products and its sequence has not yet been completely determined.

Partial Hvdroljsis of Fragments I , 3 and 5 To establish their complete sequence. fragments 1, 3 and 5 were submitted to partial T, RNase hydrolysis and the partial enzymatic digestion products were separated on 15 polyacrylamide gels (Fig. 3) followed by purification on more concentrated gels (from 18 to 24 %). Each partial digestion product was then analyzed by complete digestion with TI and pancreatic RNases. The information provided by this analysis was sufficient to establish the sequence of the smaller partial digestion products and to deduce the complete sequence of fraglnents 3 and 5 (Fig. 4). Knowing the sequence Of fragments and 5- One can deduce the sequencc of fragment 1 from the information on its oligonucleotides given in Table 2. Fragment 5 corresponds to the 5'-OH end of the fragment 1 and fragment 3 to the 3'-OH end. the two fragments being bound by A-G-A-U-G (Fig. 5). Fragment 1 will also be referred to as the "specifically encapsidated RNA fragment". Evidence for the Occurrence of a "Hidden Break" in Frugment I If the RNA extracted from the reconstituted material was loaded onto a lo';, polyacrylainide gel without 8 M urea, a major band corresponding to fragment 1 (as indicated by its oligonucleotide composition) was obtained (Fig. 6A). This material was ELII-.J . Biochem. 54 (1975)

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

139

Fig. 2. Fingerprints uf the puicrearic cznd T, rihoriucleuse digc*$/s1~ (11 fragments / (a, d } , 3 ( b , e) and 5 (c, f).Digests were fractionated by electrophoresis on cellulose acetate at pII 3.5 in the first dimension

(from right to left) and on DEA4E-cellulose paper in 7 (v;v) HCOOH in the second dimension (from top to bottom). Oligonucleotides are identified in Table 2

excised from the gel, dialyzed against 8 M urea in water and loaded onto a 10% polyacrylamide gel containing 8 M urea. Fig. 6 B shows that two new bands (1 a and 1 b) have appeared corresponding to

fragments smaller than fragment 1. When analyzed by the fingerprint methods, these fragments were found to be the two halves of fragment 1. They contain respectively 52 nucleotides for the fragment 1a and 51

Eur. J. Biochem. 54 (3975)

1 40

Sequence of a Specifically Encapsidated TMV RNA Fragment

Table 2. Molur yields qf oligonuckeolides in T, and pancreatic ribonuclease digests rffragrnent 1. 3 and 5 For a typical experiment, the radioactivity per nucleotide, estimated by liquid scintillation counting of the cxciscd spots, was respectively 300, 700 and 650 countsimin for fragments 1, 3 and 5. exp. = experimentalvalue, th. = theoreticalvalue Spot

Scquencc

Pancreatic ribonuclease products for fragment 1

exp.

5

1

th.

exp.

th.

exp.

th.

8 6 2 3 1 3 2

4.3 2.3 1.0 1.8 1.2 2.0 2.2

5 3 1 1 1 2 2

3.8

5.4 1.2

3 4 1

1.1

1

mol/chain

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 Pi2 P13 P14 Pl5 Pi 6 P17 PI8 P1V P20 P2 1 P22

Spot

U

A-C G-C A-U G-A-C A-A-1: G-A-A-C A-G-A-C G-A-A-A-C A-A -A -U G-G-C A-G-CI-C ti-U G-A-U A-G-A-U A-A-G -7J G-A-A-A-A-U G-G-U A-G-A-G-U A-A-G-G-A-G-C A-CJ

7.8 6.2 2.3 3.1 3.7 3.0 2.3 1.0 1.0 1.o 1.1 0.9 0.9 2.6 1.3 0.9 0.9 1.o 1.1 1 .0 0.9 0.9

Sequence

T, ribonuclease products for fragment

c

1

-

-

1 1 1 1

1.0

1

t 2 1 1 1 1 1 t 1 1

~~

1.1

1

-

-

1.0 1.0 -

1

1 -

1 .0 0.9 1.2 1.1

1 1 1 1

0.9 1.2 -

-

-

-

-

1.1 0.9

1 1

1.1 0.9 0.8 0.8

~

1 1 1 1

3

1 exp.

-

~

-

~

-~

1 1

-

~

-

-

0.8

1

5

th.

exp.

th.

exp.

4 3 2

3.1 2.5 2.2 3.1

3 2 2

0.9 0.9

th.

mol/chain

T1 T2 T3 T4 T5 T6 T7

'r 8

TY T10 T11 T12 Ti3 T14 T15 T16 Ti7

rig

G

c -cr

A-G C-C-G A-C-G A-A-G A-4-A-C-G A-A-C (C, j . A-C) G U-G U-A-CJ 4-U-G 4-C-U-G C-C-A-U-A-A-G A-A-A-A-U-C-A-G U-U-G U-l, -A-G ('-U-A-C (U, C) G A-U-A-A-A-U (A-A-U, U,

3)

A-A-U-A-G

3.7 2.2 2.5 0.7 2.8 0.8 0.9 0.8 1.0 2.3 1.1 1 .o 1.0 0.9 0.4 1.2 1.3 1.1

1

3 I 1 1 1 2 1 1 1 1

1.1

3

-

-

1.0

1 1 1 1

0.6 1.0 0.8

-

0.8 1.1

0.7 0.9 1.0

1

1

b u r J. Biochem. 54 (1975)

141

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

Table 3. Sequences of thc long oligonucleotides obtained from the total T I rihonucbase digests The molar yields of the products of pancreatic and U2 RNase digestion were estimated by visual inspection of the autoradiograms. The molar yields of the products of total snake venom phosphodiesterase digestion were determined by liquid scintillation counting. If a product is underlined once, it was present in two-molar yield; if underlined twice, in three or more molar yield ~~

~

~

spot

Pancreatic ribonuclease

U2 ribonuclease

TI2 TI3 Ti4 717 T18

U, G , A-C C, .4-U, A-A-G C,A-G,A-A-A-A-U U,C , G , A-C V_,A-U,A-G,&U,A-A-A-U -

A, (C, U) G G,A,C-C-A,U-A A. G, (U, C) A ??,U) A, (C, U) G A , U - A . U3-4A -~

~~

~~

a

Total snake venom phosphodiesterase

Sequence deduced

~

A-C-U-G C-C-A-U-A-A-G A-A-A-A-U-C-A-G C_. A. G C-U-A-C (U, C ) G A-U-A-A-A-U.A-A-U-A-G,A-U-A-A-A-U(A-A-U,U, (A-A-U, U2-3)b A-A-U-A-G”

C,A.U,G

v,

3)

From T, partial digest of fragment 3, it can be demonstrated that A-U is the 5’-terminal oligonucleotide of spot T18 Thcse oligonucleotides were obtained by partial pancreatic ribonuclease hqdrolysis of fragment Ti8

nucleotides for the fragment 1b. These two fragments must have been held together during the gel electrophoresis without urea probably by hydrogen bonding. These bonds were broken by the 8 M urea present during the second gel electrophoresis and the two fragments were separated. All of fragment 1 does not contain this “hidden break” as the intact sequence was found even in 8 M urea. The scission responsible for the “hidden break” presumably occurs during the initial partial digestion of TMV RNA before reconstitution. Secondury Structure of the Sequence oj Frugment 1

Fig. 3. Fractionation on 15