Resynchronization of RNA synthesis by coliphage Qβ replicase at an internal site of the RNA template

J. Mol. Biol. (1973) 76, 271-284

Resynchronization of RNA Synthesis by Coliphage Qj? Replicase at an Internal Site of the RNA Template D.

KOLAKOFSKY~,

Institut fiir

M. A. BILLETER,

H. WEBER

AND

C. WEISSMANN

der Universitdit Eiirich Ziirich, Switzerland

&lolekularbiologie

(Received 27 October 1972) In previous work Qj3 replicase has been used to synthesize labelled 5’ terminal segments of Q/3 plus or minus strands of defined length. A procedure has now been developed which allows resynchronization of Q/I replicase at an internal position and synthesis of a labelled minus-strand segment complementary to the coat cistron ribosome binding site and the intercistronic region between the A, (maturation) and the coat cistron. Resynchronization is accomplished by binding a ribosome to Q/3 RNA and allowing Qp replicase to initiate and elongate up to the ribosome, using unlabelled ribonucleoside triphosphates. The ribosome is dissociated by EDTA treatment and the EDTA is removed. The replicating complex remains functional after this treatment, and addition of labelled substrates leads to synchronized elongation. The radioactive part of the product recovered after a short elongation period with labelled substrates was shown to be complementary to the coat protein ribosome binding site.

1. Introduction The RNA-containing coliphages, such as Q/3, R17 or f2, have been intensively studied in the last few years (cf. reviews by Zinder, 1965; Weissmann & Ochoa, 1967; Lodish, 1968; Stavis & August, 1970; Weissmann et al., 1973). The RNA of these phages 1971) and their comprises 3500 to 4500 nucleotides (Sinha et al., 1965; Boedtker, nucleotide sequence is being studied in several laboratories (Adams et d., 1969; Steitz, 1969,1972; Hindley & Staples, 1969; Billeter et al., 1969a,b; Staples & Hindley, 1971; Nichols, 1970; Goodman et al., 1970; de Wachter et al., 1971; Gory et al., 1972; Adams et al., 1972; Min Jou et al., 1972). A particular approach to the problem of nucleotide sequence determination, developed in our laboratory, is based on the synchronized in vitro synthesis of Q/? RNA labelled with 32P in defined, relatively short regions (Billeter et al., 1969a,b). Synchronization is achieved by incubating Q/3 replicase with template RNA, GTP and ATP, which allows all competent enzyme molecules to initiate, and to synthesize a complementary RNA strand up to the position where the first pyrimidine nucleotide is required. Upon addition of UTP and CTP synchronous elongation ensues, and is allowed to proceed for the time necessary to give the desired chain length (10 to several hundred nucleotides). Radioactive triphosphates may be present either from the beginnning of the reaction, or may be added after elongation has proceeded to a certain extent with cold substrates to yield labelled RNA segments from internal t Present

address:

Institut

de Biologie

Molcculaire,

l&d 271

Universitir

de GenBve, Ge&ve,

Switzer-

212

E!Z’ AL.

ID. KOLAKOPSKY

regions of the RNA. These radioactive chains are then degraded to oligonucleoticles by specific RNases and analyzed by stdara sequencing techniques (Sanger et al., 1965). In addition, RNA preparations labellecl by each precursor separately give nearest-neighbour data which greatly aid the sequence analysis (Bishop et al,, 1968; Billeter et d., 1969a,b). Using these techniques with Q/3 RNA minus strands as template, the first 330 nucleotides at the 5’ region of Qp RNA were determined (Billeter et al., 1969a,b; Hindley, Billeter, Goodman t Weissmann, unpublished results). With plus strands as template 160 nucleotides of the 5’ region of minus strands were established which allowed the sequence at the 3’ region of Q/3 RNA to be deduced (Goodman et al., 1970, and unpublished results). Analysis can be pursued further in this manner, but as synthesis is extended along the RNA there is progressive loss of synchrony which results in less sharply defined labelled RNA segments. It seemed therefore desirable to develop a method of re-synchronizing RNA synthesis at some well-defined position within the RNA. In this paper we describe how this objective was attained. A,

Protein 3

1

I m >

Replicose L 0

I 1000

I

2000

I

3000

I

4000

I

FIQ. 1. A map of the C&3genome. This map is based on data from Hindley et al. (1970), Staples et al. (1971), Moore et al. (1971) and Steitz (1972). The regions of known nucleotide sequences are indicated by the narrow bars under the map. Non-coding areas are dark or hatched.

Ribosome biding to mature, native Qp RNA occurs predominantly at the beginning of the coat cistron. Figure 1 shows the map of the Q@genome and the position of the principal ribosomal binding site. In order to resynchronize RNA synthesis a ribosome was bound to Q/l RNA and the resulting complex was used as template for Q,!?replicase. As expected from earlier results (Kolakofsky & Weissmann, 1971) synthesis (carried out with non-labellecl substrates) proceeded only up to the bound ribosome. Treatment with EDTA dislodged the ribosome without dissociating the replicating complex. No nucleotide incorporation takes place in the presence of EDTA. After removal of both EDTA and the m&belled substrates RNA synthesis was allowed to resume for a short period with a-32P-labelled ribonucleosicle triphosphates. By this procedure radioactive segments of minus strands were obtained, extending from a region complementary to the beginning of the coat protein cistron towards the region complementary to the end of the A, protein cistron.

2. Materials and Methods (a) Materials Unlabelled ribonucleoside triphosphates were obtained from Schuchardt GmbH, 3H-labelled ribonucleoside triphosphates from the Radiochemical Centre (Amersham, 32P-labelled ribonucleoside monophosphates were prepared according to England).

RESYNCHRONIZATION

OF RNA

273

SYNTHESIS

Symons (1968) and converted to the triphosphates enzymically. BrUTP and BrCTP were prepared by the method of Chamberlin t Berg (1964). C&O, and CsCl were purchased from British Drug House and Merck t Co., respectively. Polyethylene sulphonate (lot no. 1993-95 U 6812) wss a gift from the Upjohn Co. (Kalamazoo, Mich.). Sodium dodecyl sulphate was recrystallized from ethanol. Pronase was purchased from Calbiochem (Los Angeles, Calif.). DEAE-cellulose (Cellex D, O-5 mequiv/g) from Calbiochem was washed with O-5 na-NaGH, O-5 M-HCl, 30% triethylamine (adjusted to pH 9.5 with COJ and finally with 2 x Tris/Na/EDTA (Tris/Na/EDTA buffer is O-1 a6-NaCl, 0.05 as-Tris*HCl (pH 7*5), 0.005 M-EDTA), and then autoclaved. (b) General prepwationa Qfl RNA, labelled or unlabelled, was prepared as described in Weissmann et al. (1968). Qp replicase wss purified as outlined earlier (Kondo et al., 1970). The replicase preparations were devoid of RNsse activity, had specitic activities between 2000 and 4000 units/ mg (1 unit of activity corresponds to the incorporation of 1 nmol of GMP in 20 min, under the assay conditions described in Kondo t Weissmann (1972a)), and were about 80% pure as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulphate, by the criteria of Kamen (1970) and Kondo et al. (1970). Host factor was prepared as described by Kondo & Weissmann (19725). The preparation of ribosomes from Esc~erichia coli Q13 and fMet-tRNA has been described (Kolakofsky & Weissmann, 1971). One Aaao unit of ribosomes bound 10 pmol of fMet-tRNA in response to Q,3 RNA, without additional initiation factors. (c) Preparation

of ribosome-Q/3

RNA complex

The binding mixture contained in a total volume of 0.9 ml, 60 mm-Tris*HCl (pH 7*5), 120 mM-m,Cl, 8 m&r-Mg&, 08 ma6-dithiothreitol, 0.5 m&t-GTP, 4% glycerol, 32 0.D.2ao units of ribosomes, 52 pg of unfractionated fMet-tRNA (3H-labelled at 1500 cts/min/pmol or non-radioactive) and 700 pg of Qp RNA (3aP-labelled at 1650 cts/min/pg or unlabelled). After 10 min at 37”C, the mixture was layered on three 10% to 30% linear sucrose gradients (5 ml) containing 10 ma-Tris*HCl (pH 7*5), 10 mM-MgCl,, 50 mm-NH&l and 0.5 mMdithiothreitol, and centrifuged for 70 mm at 60,000 revs/min (4°C) in an SW66 Spinco rotor. Fractions (24) were collected, the aaP profile was determined by Cerenkov counting (or the absorbancy profile at 260 nm was measured) and the fractions corresponding to the 70 S peak (usually about I.5 ml from 3 gradients) were pooled. 100 d of this solution contained, in a typical experiment, 2.5 pmol of fMet-tRNA and 2.7 pg of Qj3 RNA in a ribosome complex, as determined by adsorption to Millipore filters (Nirenberg & Leder, 1964). For the preparation of larger quantities of the complex, up to 4.2 ml of binding mixture were used. The 70 S complex was then isolated after centrifugation for 3 h at 38,000 revs/ min in an SW41 Spinco rotor. The maximum ratio of sample volume to gradient volume was 1:17. (d) Resynchronization

of minus-strand synthesis ad the coat &&on binding followed by short-time synthesis with radioactive sub&ate

site

To 1 ml of 70 S complex the following components were added (in a negligible volume) : TriseHCl (final concn 60 mm), MgCl, (14 mu), GTP and ATP (0.4 mM each), 30 to 40 units of Qj3 replicate (about 10 pg of protein) and a saturating amount of host factor (about 10 pg of the partially purified protein). Initiation was allowed to proceed for 6 min at 37°C and a mixture of CTP, UTP and polyethylene sulphonate (in 30 ~1) was added to give final concentrations of 0.4 mm, 0.4 mM and 0.04 mg/ml, respectively. After 6 min at 37°C the mixture was chilled in an ice bath and 0.1 vol. of 0.5 M-EDTA was added. The preparation was passed through a column of Sephadex G50 (fine) equilibrated with 30 rnas-Tris*HCl, pH 7.5, at 4°C (the ratio of sample to column volume was 1: 10). The excluded material was located by measuring the absorbance at 260 nm and the peak fractions were pooled to give a volume not larger than that of the sample applied to the column. After addition of polyethylene sulphonate (0.04 mg/ml) the mixture was adjusted to 20°C

374

ET AL.

D. KOLAKOFSKY

and poured rapidly into a centrifuge tube containing the four triphosphates (either all or only one labelled) to give a final concentration of 0.01 to 0.1 mM, Tris*HCl, pH 7.5 (final concn, 50 mM) and MgCl, (final concn, 12 mM). After 5 to 50 s (depending on the concentrations of triphosphates used and the desired length of the labelled segment) 2-5 vol. absolute ethanol at - 20°C were rapidly mixed in. After 5 min the precipitate was sedimented by centrifugation for 10 min at 10,000 revs/min and the pellet was dissolved in 100 mM-EDTA (0.1 the incubation volume) and passed through a small column of Sephadex G50 (bed volume, 10 times the sample volume), equilibrated with 20 mMTris.HCl, pH 7.5. The excluded radioactivity was located by Cerenkov counting, the corresponding fractions were pooled and incubated for 10 min at 37°C with sodium dodecyl sulphate (0.5%) and pronase (0.2 mg/ml, preincubated at 20 mg/ml in 150 mm\l-EDTA for 2 h at 37°C and stored frozen). The RNA was twice extracted with phenol and precipitated with 2 vol. ethanol in the presence of 0.1 M-N&I, 0.05 M-Tris*HCl (pH 7.5), 0.005 nr-EDTA.

(e) Pur$cdion

of the product

The precipitate, containing up to 3 mg of RNA, was dissolved in O-5 ml of Tris/Na/EDTA buffer and passed through a column (1.2 cm X 40 cm) of Sepharose 4B (Pharmacia Fine Chemicals) equilibrated in Tris/Na/EDTA at a flow rate of l-5 ml/h. This procedure separates the double-stranded 32P-labelled product (excluded peak) from the large amount of ribosomal RNA (included peak) (Erikson & Gordon, 1966). The excluded fractions (containing at the most 40 pg of RNA) were pooled. The product thus obtained was frequently suitable for sequence analysis without further purification (see below). After several precipitations with ethanol it was dissolved in 10 pl of water and denatured in a capillary tube for 2 min in a boiling water bath and quickly cooled. One sample was immediately digested with ribonuclease T, and another with pancreatic ribonucleaae, and the digestion products were separated by 2-dimensional electrophoresis, as described by Sanger et al. (1965). When the binding of ribosomes had occurred not only at the beginning of the coat protein cistron but also at the replicas0 cistron (see Discussion), aa shown by the appearance of two discrete peaks of product on zonal sedimentation analysis, the product was heat-denatured and the labelled RNA resolved on a composite 0.5% agarose/3+%

polyacrylamide

gel slab in Tris/borate/EDTA

buffer as described (Dingman

& Peacock,

1968; Peacock & Dingman, 1968). Electrophoresis was overnight at about 6 V/cm. The two separated bands were located by autoradiography and cut out. The gel strips were homogenized in 1.5 ml of 2 x Tris/Na/EDTA containing 20 pg of ribosomal RNA, in a motor-driven homogenizer. The slurry wm centrifuged, the supernatant fraction collected and the pellet extracted twice more, as described above. The combined supernatant fractions were pumped at a rate of about 0.5 mg/ml through a 0.15 ml-column of DEAEcellulose (washed as described in the Materials and Methods section) and after washing the columns with 1 ml of 6% triethylamine bicarbonate (pH 9.5) the RNA was eluted with 3 ml of 30% triethylamine bicarbonate (pH 9.5). The extract, containing more than 80% of tho radioactivity initially in the gel slice, was evaporated several times to dryness from water in an evaporating apparatus (Evapomix Buchler). The residue was transferred to a small centrifuge tube in a total volume of 0.5 ml Tris/Na/EDTA, and the RNA was precipitated with 1 ml of ethanol. After standing the tube overnight at -20°C the RNA was collected by ccntrifugation and processed for enzymic digestion essentially as described above, but omitting the heat denaturation step.

3. Results (a) Binding

of ribosomes to Q/3 RNA

blocks chain elongation

by Q/3 replicate

Rihosomes carrying protein synthesis initiation factors were bound to Q/3 RNA using fMet-tRNA (labelled with 3H) and the resulting 70 S complex was isolated by sucrose gradient eentrifugation. The complex was incubated with Q/? replicase, nucleoside triphosphates and host factor under standard conditions of RNA synthesis. It had been shown earlier that after 15 minutes at 37°C more than 85% of the

(u!w/sls)

~Pwoo!pDJ

d,,

s

8

I

I’

(U!U/

SIC’)

alqnlosu!-pyv

r(l!A!lSDO!POl

HE

a(q”(oS”l

-p!3v

276

D. KOLAKOFSKY

ET AL.

3H-labelled fMet-tRNA remained bound to the ribosomes, whether or not Q/3 replicase and host factors were present. Thus, replicase does not dislodge bound ribosomes from the template under conditions of RNA synthesis (Kolakofsky & Weissmann, 1971). Moreover, the 70 S complex proved to be a relatively inefficient template for Qp replicase; in a five-minute incubation nucleotide incorporation was about 15% of a control containing free Q/3 RNA in addition to the 70 S complex (Kolakofsky & Weissmann, 1971). The product of the restricted RNA synthesis resulting from the use of the 70 S ribosomal complex as template for Qfi replicase contained very little full-length 30 S RNA and sedimented predominantly around 25 S. The product of the control reaction, in which a mixture of free Q/3 RNA and 70 S complex was used as template, yielded mostly full-length RNA (cf. Fig. 2(a) in Kolakofsky t Weissmann, 1971). The formation of a small amount of 30 S RNA was probably due to traces of free Qfl RNA, arising from dissociation of the complex, which were replicated to detectable amounts within a few minutes of incubation with Qp replicase. In order to avoid this complication, Q/3 replicase and the ribosome-Q/l RNA complex were allowed to initiate RNA synthesis with GTP and ATP, and then an inhibitor of initiation, polyethylene sulfonate (Kondo & Weissmann, 19723), was added before starting elongation with UTP and CTP. Under these conditions no detectable 30 S product was formed (Fig. 2(a)). As mentioned earlier, ribosomes bind to native Q/3RNA predominantly at the initiation site of the coat cistron, i.e. between the 1100th and 1400th nucleotide from the 5’ end of the Q/3 RNA (which comprises a total of about 4500 nucleotides (Boedtker, 1971)). If synthesis of the minus strand proceeded only up to this position, its length would correspond to about 3250 nucleotides and its .~sa,~value would be 25.5 S, as calculated by the relation 8,/S, = (X,/iVJk with 8, = 30.5, M, = 4500 and k = O-56 (Studier, 1965). This agrees well with the value of 25 S found. (b) EDTA-treatment of the ribosome-blocked replicating complex removes the ribosome without dislodging replicase and the nascent RNA chain Treatment of the ribosome-Q/3 complex RNA with EDTA releases the ribosome from the viral RNA, as shown by sucrose gradient analyses of complexes containing csP-labelled RNA (data not shown). In order to establish whether it was possible to remove the ribosome from a ribosome-blocked replicating complex by EDTA treatment without dislodging replicase and the nascent RNA strand from the template the following experiment was devised. A ribosome-Q/3 RNA complex was incubated with replicase, factor, GTP and ATP to give the initiation complex; polyethylene sulphonate was then added to prevent further initiation. Chain elongation was started by addition of UTP and CTP, and after five minutes of incubation one-half of the mixture was removed for analysis. Sedimentation through a sucrose gradient showed that the 3H-labelled RNA sedimented at about 25 S (Fig. 2(a)). The other half of the preparation was treated with EDTA to detach the ribosomes, and EDTA as well as the substrates were then removed by chromatography through Sephadex. Magnesium ions, polyethylene sulphonate and the four standard triphosphates including a-3aP-labelled GTP were added and incubation was carried out at 37°C for a further three minutes to allow completion of the minus strands. The sedimentation profile (Fig. 2(b)) of the final product showed that about 40% of the 3H radioactivity had been transferred from the 25 S to the 30 S position, and virtually all of the 32P

RESYNCHRONIZATION

OF RNA

Polyethylene

SYNTHESIS

277

sulphonate

ATP,[~HIGTP BrUTP,

BrCTP

bo

32P GTP,ATP,UTP,CTP

..........

@ ................ 32P, light Sucrose gradient

-o CsCl density gradient

FIG,. 3. Two-stage synthesis of a minus strand labelled sequent.ially with heavy and light nucleotides (sclheme). In the first stage of synthesis a ribosome-Q/3 RNA complex is used as template and synthesis is carried out up to the ribosome, using ATP, [3H]GTP, BrUTP and BrCTP to yield a densityrmd 3H-labelled minus strand segment. (Polyethylene sulphonete is added aftar initiation to inaotivate free repliease and thereby prevent subsequent rein&i&ion, cf. Kondo & Weissmann (1972b).) The ribosome is dislodged by EDTA treatment, EDTA and substrates are removed by Sephadex ohromatography. On subsequent incubation with the four standard triphosphstes, including [3sP]GTP, the heavy, sH-labelled minus-strand segment is completed with a light, 32P.labelled segment. The graphs on the right- and left-hand side of the scheme show the idealized profiles expected for a CsCl density gradient equilibrium oentrifugetion and a sucrose gradient velocity sedimentation, respectively.

radioactivity was found in the latter position. Since polyethylene sulphonate completely inhibits initiation of new chains, these findings strongly suggest that minus strands that had been started during the first phase of synthesis were completed during the second stage of the reaction, after removal of the ribosomes. To obtain definitive proof for this conclusion the experiment outlined in Figure 3 was carried out. For the first phase of minus-strand synthesis, ATP, [3H]GTP, BrCTP and BrUTP were used as substrates. A sample of the resulting product was denatured and banded in a Cs,SO,/ CsCl gradient (Fig. 4(a)) ; as expected, the 3H and density-labelled minus strand had a much greater buoyant density than natural Qfl RNA (plus and minus strands have almost the same buoyant density; Pallet et al., 1967). The bulk of the complex was treated with EDTA, chromatographed through Sephadex and incubated with the four standard triphosphates, including [a-32P]GTP. The full-length virus RNA was isolated by sucrose gradient centrifugation, denatured and then banded in a density gradient as above; both the 32P and the 3H radioactivity banded in the same position with a buoyant density of 1.91 g/cm3 (Fig. 4(b)). The product was thus denser than light RNA, but substantially less dense than RNA labelled throughout with 10

Fraction no.

C

IOC

2oc

FIG. 4. Two-stage synthesis of a minus-strand density hybrid: isopycnic Synthesis was carried out as described in the legend to Fig. 2, except that in the first stage CTP and the second stage [a-32P]GTP (262,000 cts/min/nmol) was used as labelled precursor. Samples were taken titer the first stage (A) and second stage (B) of synthesis and centrifuged through of A (25 S) and the peak fractions of B corresponding to full-length RNA (30.6 S) were precipitated with denatured and centrifuged in a CsCl/Cs,SO,/formaldehyde gradient as described by Lozeron t Szybalski

(a)

V’I 86

Product A

by BrCTP and BrUTP

and in a sucrose gradient (cf. Fig. 2). The peak fractions ethanol. The RNA w&s dissolved in water, heat (1966).

gradient analysis. UTP were replaced

Product B

RESYNCHRONIZATION

OF RNA

SYNTHESIS

279

BrCTP and BrUTP. These analyses conclusively show that minus strands begun in the first phase of synthesis (with heavy, 3H-labelled substrates) were completed (with light, 32P-labelled substrates) in the second phase, after removal of the ribosome, to give heavy-light end-to-end hybrids. Resynchronization of RNA synthesis at the position of the ribosomal binding site thus became feasible. (c) The labelled RNA synthesized during a short-time incubation after removal of the ribosome from the Q/3 RNA-ribosome complex wrrespomh to a homogeneous segment of the Q/l minus strand The homogeneity of an RNA preparation of known chain length can be estimated from the complexity of the fingerprint obtained after its digestion with RNase T,. Thus, a homogeneously labelled RNA preparation of about 25 mole% G oontent and a random sequence, and with a chain length of 50, would be expected to yield about 12 different fragments; a mixture of two species of this same chain length would give rise to about 24, a mixture of 10 species to around 86 different fragments. 3’ ‘&,

5’

i-

8

m a t

fT$icase,

factor

GTP

ATP,UTP.CTP

FIG. 5. Resynchronization of minus-strand synthesis at the ribosomal binding site end synthesis of a short’, radioactive segment (scheme). The Qfl RNA-ribosome complex was incubated with &p replicase, factor, ATP and GTP to give an early replicating complex. Polyethylene sulphonate was added to ineativate any free enzyme and thereby prevent initiation during the later phase of the experiment. Elongation was started by adding UTP and CTP (’m some experiments [3H]UTP was used to monitor the f&t phase of synthesis). After 5 min at 37°C EDTA was added to remove the ribosome from the complex. EDTA and substrates were removed by Sephadex chromatography. The complex was then incubated with the four standard triphosphates at 0.01 mM, of which one or all were a-labelled with 32P at high specific activity, for 60 to 100 s at 2O”C, to allow synthesis of a labelled segment of about 60 to 100 nucleotides in length.

4

6,

ApU;;

ApApApGp(O.7); CP(l.7) a

ApApApGh

Ap+C;(l);

iifi&iGpfl);

4

ATP

GTP

NTP

UTP

CTP

ATP

GTP

ApCp(1);

Gp(1)

Gp(1)

TABLE

I

(CP)AP,

product

-

(Up)zGp:

-

-

-

AP

Deduction

digestion

l

*

APCPCPCPAPAPA+PGPVJI ACCCAAAGr U 1

APCPCPC~APAPAPGPWI

+

APCPCPCPAPAPAPGPIYI

(A~~C~~)!CP)(CP)APAPAPGP~U~

(APCP)(CP)(CP)APAPAPG~

CCAUG[Al

CPCPAPU;GP[AI

CpC;ApUpG;[A\

C~PAPUPGP

CPCP&JPGP

CPCPAPUPGP

A~UPC~APAPAPUPU;GP AUCAAAUUG

A;UpCpApApA;U;UpGp

APAPAPUPCPAPUPUPGP o= APUPC~APAPAPUPUPGIJ

UPC~UPCPUPAPAPUPUPUPU;GPFI UCUCUAAUUUUG [C]

UPCPUP&A;APUPUPUPUPGPFI

U;CpU;CpUpApApUpUpUpUpG;[C]

(CPZUPZ)APAPUPUPUPUPGP

from T, ribonuclease

(UP), - .GP: AP

UPGP -

Wp,TJp)Ap;

(CxjaU~dAp;

Ua digestion

oligonucleotides

as the one described in the legend to Plate I were carried out with either ATP, UTP, CTP or GTP ax the only a-32P-l&belled of the other three unlabelled substrates) or with all four triphosphaLes labelled. The products were processed end digested Materials and Methods. The digestion products were separated, isolated and further analyzed by degrade&m with pancreatic obtained were hydrolyzed to mononucleotides with T, RNrtse (Hiramaru Uz RNase (Arima et (II., 196s). The oligonucleotides by *). The values in parent.heses give the relative number of moles of oligonucleotides reoovered, the 321) lehel (indicated underlined oligonucleotide.

Cpf(1.3)

Cp(2.2);

C;(l)

A%P

UTP

CTP

G;;

A~Up(l.1);

U;:

GTP

; A;Up

6 (2)

I

product

of the four

&Up(l);

(2);

Gp(l)

u;(l)

Up(l);

NTP

Ap ApA;U;

WO43;

UTP

NTP

C;

(2)

(1);

(O-9); UP(6.1)

digestion

sequences

ApApApUp(OW;

cp”cl,:r.T;;

ZGJpU):

ATP

GTP

ApA%

CTP

cpw

APAPUP

Pancre&ic

UTP

NTP

label

Labeliiug experiments such triphosphate (in the presence with T1 RN&se &s described in RN&se (Sanger et al., 1966), or et al., 1966) in order to localize assigning the value of 1 to the

T20

TCiO

T26

Tl

a.=P

Nucleotide

. . . , . . UGAAA

Ribosome

\

Tl

......

\ C......

I

site, coat cistron

CAAAAUUAGAGA

binding

GAAACCCAGUUAAACUAGUACCGUTJUUAAUCUCU \ I\ /\ I\T26 T60 T20

CUUCJGGGU

site

I CAAUUUGAUCAUGG

Replicate-protected

plus strand minus strand

3’ 6’

FIO. 6. Nucleotide sequence at the coat cistron ribosome binding site and the oligonucleotides isolated from the RNA segment pulse-labelled after resynchronizetion. The nucleotide sequence of the ribosome binding site at the coat cistron was determined by Hindley t Staples (1969), that of the replicase binding site by Weber et al. (1972). Oligonucleotides Tl, T20, T26 and TSO (Table 1) were recovered as the only products following T, RN&se digestion of the minus.atrand segment l&belled after resyncbronization at the coat cistron riborrome binding site as described in Plate I. (The order of the oligonucleotides was not established experimentally.)

3’

6’

I

T26

-i-~~

(b)

0 T50 T20

-‘p_----

Orlgln --<

and synthesis PLATE I. (a) Fingerprint of a short minus-strand segment labclled after reaynchronization at the ribosomal binding site. &synchronization of the product, at IO* cts/min/nmol). as well as purification (for 50 s at 20°C) in the presence of the four nuoleoside triphosphatcs (0.01 m&r, all a- 3ZP-labelled wore carried out as outlinccl schematicallv in the legend to Fig. 5. The detailed experimental procedure is described in Materials and Methods. The purified. “2P-labelled product (about 5 x 105 cts/m’in) was tligested with RN&e T, and analysed by two-dimensional electrophorcsi* (first dimension, cellulose acetate acid) as described by Sanger et nl. (1965). at $3 3.5: second dimension, DE.%E-paper in 7 Oh formic (b) Tracing of (a).

(a)

TI

-a-.

Cellulose acetate , pti 3.5

282

D. KOLAKOFSKY

EI’

AL.

We prepared a ribosome-Q/3 RNA complex and utilized this as template for minusstrand synthesis as described above, except that we used unlabelled substrates (cf. scheme, Fig. 5). After detachment of the ribosomes with EDTA and reisolation of the replicating complex, synthesis was restarted by adding, at a very low concentration, the four standard nucleoside t,riphosphates, all labelled with 32P in the CLposition. After 50 seconds at 20°C synthesis was arrested, the RNA was isolated, subjected to digestion by T, RNase and the fragments were separated by two-dimensional electrophoresis. The resulting fingerprint (Plate I(a)) shows four well-defined spots. The elongation rate at 20°C under the conditions of the experiment is about one nucleotide per second (Billeter, unpublished results). Incubation for 50 seconds should therefore allow the synthesis of a segment of around 50 nucleotides chain length, which would be expected to give about 12 spots on the fingerprint under the assumptions stated above. The fragment synthesized in fact proved to contain only 34 nucleotides and 12% G. The nucleotide sequences of the four T, oligonucleotides, Tl, T20, T26 and T50, isolated from the short product labelled after resynchronization were determined (cf. Table 1). It is evident (Fig. 6) that these oligonucleotides are derived from the sequence complementary to the ribosome binding site at the beginning of the coat cistron (Hindley & Staples, 1969) and the preceding intercistronic region (Weber et al., 1972). One may note that incorporation of radioactive substrate into the minus strand after synchronisation started precisely at the region complementary to the 3’ terminus of the QF RNA segment which is protected against RNase by the ribosome in the binding complex. This indicates that Q/J replicase had run up to the immediate vicinity of the ribosome during the first phase of synthesis. Since the coat protein cistron is preceded by the A, cistron (Staples et al., 1971), the labelled minus-strand segment extends from the region complementary to the beginning of the coat protein cistron towards the region complementary to the end of the A, cistron.

4. Discussion The procedure for resynchronizing Q/l RNA synthesis is based on three phenomena. (1) Ribosomes can be bound to a specific site on Qp RNA. (2) Qj3 replicase can use the ribosome-Q/3 RNA complex as template up to the binding site of the ribosome but is unable to dislodge the ribosome. (3) Treatment of a ribosome-blocked replicating complex with EDTA leads to release of the ribosome, but retention of replicase and the nascent RNA strand in an active form. No nucleotide incorporation takes place in an excess of EDTA over magnesium ions. While ribosomes bind predominantly to the beginning of the coat cistron on native Qfl RNA, we have occasionally obtained batches of Q,9 RNA to which a substantial proportion of the ribosomes bound at the beginning of the replicase cistron. No difference was noted in the sedimentation behaviour of these different Qj3 RNA preparations, all of which were prepared by similar methods. It is possible to favour binding at the replicase cistron to some extent by heating the RNA to 5O”C, or higher, before use. Somewhat similar findings were reported on R17 RNA by Fukami & Imahori (1971). When ribosomes bind at the coat as well as at the replicase cistron, resynchronization of RNA synthesis as described in this paper yields two products, one sedimenting at 25 S, and the other at about 19.5 S. The two products can easily be separated by acrylamide gel electrophoresis or sucrose gradient centrifugation and analysed separately. The labelled segment of the shorter product has

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been shown to be complementary to the beginning of the replicase cistron (Billeter, unpublished results). The sequential labelling techniques described earlier (Billeter et al., 1969a,b) should permit us to deduce the sequence of about 400 to 500 nucleotides, possibly more, at each of the two ends of Qp RNA; the resynchronization procedure at the beginning of the coat and replicase cistron is expected to yield the sequence of some 200 additional nucleotides in the interior of the molecule. We thank Mr Werner Romer, Miss Claire de Tscharner and Mrs Libuse Gross for their valuable technical assistance. This work was supported by grants from the Schweizerische Nationalfonds (no. 5272) and the Jane Coffin Childs Fund (no. 243). One of us (D. K.) was supported by an EMBO Fellowship. REFERENCES Adams, J. M., Jeppesen, P. G. N., Sanger, F. & Barrell, B. G. (1969). Nature, 223, 1009. Adams, J. M., Spahr, P. F. & Cory, S. (1972). Biochemiatq, 11, 976. Arima, T., Uchida, T. & Egami, F. (1968). B&hem. J. 106, 609. Billeter, M. A., Dahlberg, J. E., Goodman, H. M., Hindley, J. & Weissmann, C. (1969a). Cold Spr. Harb. Symp. Quant. Biol. 34, 635. Billeter, M. A., Dahlberg, J. E., Goodman, H. M., Hindley, J. & Weissmann, C. (1969b). Nature, 224, 1083. Bishop, D. H. L., Mills, D. R. & Spiegelman, S. (1968). Biochemistry, 7, 3744. Boedtker, H. (1971). B&him. Biophye. Acta, 240, 448. Chamberlin, M. & Berg, P. (1964). J. Mol. Biol. 8, 297. Gory, S., Adams, J. M., Spahr, P. F. & Rensing, U. (1972). J. Mol. Biol. 63, 41. Dingman, C. W. & Peacock, A. C. (1968). Biochemistry, 7, 659. Erikson, R. L. & Gordon, J. A. (1966). B&hem. Biophys. Res. Commun. 23, 422. Fukami, H. & Imahori, K. (1971). Proc. Nat. Acud. Sci., Wash. 68, 570. Goodman, H. M., Billeter, M. A., Hindley, J. & Weissmann, C. (1970). Proc. Nat. Acad. Sci., Wash. 67, 921. Hindley, J. & Staples, D. H. (1969). Nature, 224, 964. Hindley, J., Staples, D. H., Billeter, M. A. & Weissmann, C. (1970). Proc. Nat. Acad. Sci., Wash. 67, 1180. Hiramaru, M., Uchida, T. & Egami, F. (1966). Analyt. B&hem. 17, 135. Kamen, R. (1970). Nature, 228, 527. Kolakofsky, D. & Weissmann, C. (1971). Nature New Biol. 231, 42. Kondo, M. & Weissmann, C. (1972a). Eur. J. Biochem. 24, 530. Kondo, M. & Weissmann, C. (1972b). B&him. Biophys. Acta, 259, 41. Kondo, M., Gallerani, R. & Weissmann, C. (1970). Nature, 228, 525. Lodish, H. F. (1968). Proc. Biophys. Mol. Biol. 18, 285. Lozeron, H. & Szybalski, W. (1966). Biochem. Biophye. Res. Gommun. 23, 612. Min Jou, W., Haegeman, G., Ysebaert, M. & Fiers, W. (1972). Nature, 237, 82. Moore, C., Farron, F., Bohnert, D. & Weissmann, C. (1971). Nature New Biol. 234, 204. Nichols, J. L. (1970). Nature, 225, 147. Nirenberg, M. & Leder, P. (1964). Science, 145, 1399. Peacock, A. C. & Dingman, C. W. (1968). Biochemistry, 7, 668. Pollet, R., Knolle, P. & Weissmann, C. (1967). Proc. Nat. Acad. Sci., Wash. 58, 766. Sanger, F., Brownlee, G. G. & Barrel& B. G. (1965). J. Mol. BioZ. 13, 373. Sinha, N. K., Fujimura, R. K. & Kaesberg, P. (1965). J. Mol. BioZ. 11, 84. Staples, D. H. & Hindley, J. (1971). Nature New BioZ. 234, 211. Staples, D. H., Hindley, J., Billeter, M. A. & Weissmann, C. (1971). Nature New BioZ. 234, 202. Stavis, R. L. & August, J. T. (1970). Ann. Rev. Biochem. 39, 527. Steitz, J. A. (1969). Nature, 224, 957. Steitz, .J. A. (1972). Nature New BioZ. 236, 71.

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