RNA-dependent RNA

(1)

Proc. Nat.Acad. Sci.USA

Vol. 72, No. 7, pp. 2640-2643, July 1975 Biochemistry

Minimal requirements for template recognition by bacteriophage Qft

replicase: Approach to general RNA-dependent RNA synthesis

(protein-nucleicacid interaction/oligonucleotides/RNA replication) BERND KUPPERS AND MANFRED SUMPER

Max-Planck-Institut fur Biophysikalische Chemie,34Gbttingen-Nikolausberg,WestGermany

Communicated byManfredEigen, May5,1975

ABSTRACT Any oligo- orpolynucleotide able to offer a C-C-C-sequence at the 3'-terminus and a second C-CC-se- quenceinadefinedstericpositionto

Qft

replicase is an effi- cient template. Corresponding chemical modifications con- vertnon-templateRNAstotemplate RNAs.

ThesmallEscherichia colh bacteriophage

Qfl

inducesan enzyme, Qf3 replicase, which isresponsible for thereplication of the phageRNA. The enzymeconsists of onevirus-speci- fiedpolypeptide (1,2)and three hostpolypeptides. Thehost proteinsare the proteinsynthesis elongation factors Tuand

T.

(3)and theribosomalproteinS1 (4).

Thephagereplicaseshowsaveryhigh templatespecifici- tyfor thecomplementary plus(virion)and minus strandsof the homologous viral RNA (5). Unrelated viral RNAs and most other naturally occurring RNAs do not serve as tem-

plates. Despiteits capacity for discriminatingbetween

QWl-

specific RNAs and all other naturally occurring RNAs,

Qf3

replicaseaccepts poly(C)and C-containingrandomcopoly- mers (6) aswell asa varietyofsocalled "6S" RNAs(7). At firstglance,thisfactseemstobeaparadoxicalone.

The minimal requirements forRNAtemplaterecognition by Qi3 replicase are the subject of this paper. We demon- stratethattwoclusters ofcytidineresidues in adefinedste- ricposition trigger theinitiationofRNAsynthesis by

Q#3

replicase.

MATERIALS AND METHODS

Isolation of Phage QBReplicase.Phage

Qfl

replicasewas

purifiedandassayedasdescribedbyKamen(8).

Nucleotides. -y-32P-Labeled ribonucleoside triphosphates were prepared by the method of Glynn and Chappel (9).

The other labeled ribonucleoside triphosphates were pur- chased from AmershamBuchler,Braunschweig.

Primer-Dependent Polynucleotide Phosphorylase. Con- version of commercial polynucleotide phosphorylase from Micrococcus luteus

(Boehringer

Mannheim

GmbH,

Mannheim) to

oligonucleotide

primer

dependence

was

achievedby treatment with N-ethylmaleimidein the pres- enceof1 Mguanidine hydrochlorideasdescribedbyLeten- dre and Singer (10). The

resulting

enzyme preparationwas

used directly for the elongation reactions ofoligo- and po- lynucleotidesdescribed below, without removal of the gua- nidinehydrochloride.

Preparation of Homopolymers. Poly(U), poly(C), and poly(A)were

synthesized

from the

corresponding

nucleoside diphosphates

(Waldhof, Mannheim)

using

purified polynu-

cleotide

phosphorylase

isolated fromE.coliK 12Hfr

(11).

Preparationof(Cp)nCOligomers.

Poly(C)

was

degraded

toa mixtureofoligomersbylimitedhydrolysiswithpiperi- dine(6-8min at1000 in 10%

piperidine) (12).

The

oligom-

ers werefractionatedon aQAE-SephadexA-25column (1.5

X70cm) by elution with a linear gradient, 0.3-0.7 M

NaCI,

in 0.05 M Tris-HCl, pH 7.5 (one 2000 ml reservoir of each buffer). Thisgradientensures the complete resolution of the oligomers up to

(Cp)1s,

whereas the oligomers (Cp)14 to (Cp)19wereonlypartially resolved. The peak fractions were desaltedby SephadexG-10filtration. The 3'-terminalphos- phates were removed by digestion with human semen phosphatase (kindly provided by Dr. Biebricher).

(Ap)nA

and

(Up)nU

oligomers were prepared by the same method.

Preparation of

(UpM(Cp).C

^and

(Ap)(Cp).C

Oligomers.

Primer-dependent polynucleotide phosphorylase (see above) was used to add a block of C-residues to each oligonucleotide primer (Up)6Uor(Ap)6A. The reaction conditions used wereapproximately those of Martin et al. (13). A typical incubation mixture contained in 3 ml: 0.2 M glycine buffer (pH 9.2), 30 mM CDP, 0.6 M NaCl, 5 mM

Mg++,

about 200 A260 units oligonucleotide, and 0.7 mg of polynucleotide phosphorylase. Incubation was at

370

for 4-6 hr. The block copolymer products were fractionated on QAE-Sephadex A-25columns as described above, after the addition of oligonucleotide primer as an internal marker. Although the peaks could usually be identified simply by counting from the primer peak the identification was confirmed by com- pletealkaline hydrolysis of a portion of each of the first several peaks and determination of the Up (or Ap):Cp:C ratio (14).

Preparation of

(Cp)4(Up)5(Cp)C

Oligomers.

(Cp)4(Up)4U was obtained by adding a block of U-residues tothe primer(Cp)sCand separating the products on a QAE- SephadexA-25column asdescribedabove. For the addition of C-residuestothe primeroligonucleotide(Cp)4(Up)4Uand the subsequentseparationand identification of the products

(Cp)4(Up)5(Cp)nC,

theproceduresdescribed above were fol- lowed.

Preparation of Polynucleotides

(Ap),.,(Cp).C

and

(Up),(Cp)nC

^(m ^>>^n). Poly(A) orpoly(U) were treated with semen phosphatase to remove any terminal 3'-phosphates present. Then a block of C-residues was added by using primer-dependent polynucleotide phosphorylase. The incu- bationmixturecontainedin100,ul:0.1 MTris-HCl(pH8.2), 30 mM CDP, 10 mM

Mg++,

80 ,ugof poly(A) [orpoly(U)]

and 20 ,ug of polynucleotide phosphorylase. The reaction was run at

370

for3-12hrand then stopped by the addition of sodiumdodecylsulfate andadropofchloroform. The po- lynucleotideswereisolatedby chromatographyonSephadex G-50columns.

RESULTS

In addition to

QO3-specific

^{RNAs, QB} replicase accepts poly(C)and C-richcopolymers astemplates. Evidently the modeltemplatesalso havetofulfill all requirements forini- 2640

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Proc.Nat. Acad. Sci. USA 72 (1975) 2641

CG C U A UA

UA UG U U A UA

GC UA G-C ^C C UA

G-C

GGC

^G-C ^U-A ^A ^C

Gg Gl Gg G0 G;

GU GC GC CG C

A A G G C C C-G A-U

A AA CU G A A-U

A U U-A

UC G U

G C

UC

1 2 3 4 5

C'.

C C C CC

C

E

0CL w c:

0c:

0z 0.I--

0.

6000- 5000- 4000- 3000- 2000 1000

6

/ /

S;_/ o a aIIa IaI

a-w- - _& -

246 8 10 12 1,4 1,6 18 2

FIG. 1. Nucleotidesequencesof the 3'-termini of RNAs acting

astemplates for Qfi replicase.1:Qua(-)strand(15);2:Midivariant

(+) strand (16); 3: Midivariant (-)strand (16);4and 5:(+) and

(-)strand ofa"6S" RNA(W.Schaffner and C.Weissmann,per-

sonalcommunication); 6: Poly (C).

tiationof RNAsynthesis. The 3-ends of all templateRNAs sequenced^sofarterminate withasequenceofatleast three C-residues(Fig. 1).Achemicalmodification of this3'-termi- nalC-C-C-sequence leadstoaloss oftemplateactivity (17), indicating the importance of this C-cluster. Several other viral RNAs, such asthose ofphagesMS2, f2, and R17, and tobaccomosaic virus, whichalso-terminatewitha C-cluster at the 3-end, are inactive as templates. Therefore this^se-

quencecannot be theonlyrequirementfortemplaterecog-

nition by Qfl-replicase. Consequently one (or more) additional nucleotide sequences mustbe involved in theinitia- tion mechanism. Taking into account the poly(C) activity, this additional requirement can only be fulfilled byC-nucleotides.

Examination of template RNAs of known sequence re-

veals a striking feature. As shown in Fig. 1, all sequences

havein common aC-cluster ata defineddistance from the '-terminus. In ordertodemonstrate theimportanceof this internalC-C-C-sequence inthe recognitionprocess wepre-

pared several oligonucleotides with defined sequencesand investigatedtheirtemplateactivity.

(Cp)nC-ofigonucleotides

Using the idea oftwoC-clusterscooperatingin therecogni- tion process, one can predict theminimum chain length of C-oligomersactingas templates. As canbe estimated from Fig. 1,(Cp)lsCshould be thiscriticalchainlength.Intheex-

periment of Fig. 2 the oligo(C)s ranging from CpC to

(Cp)18C were assayed for template activity. No activity is observed for the oligonucleotides up to the chain length 13 (curve I). Withinthelimited rangeof(Cp)IsCto (Cp)17Ca

template activity isreached comparableto that of poly(C).

Athigher oligonucleotide concentration a limited GMP-in- corporationdirectedbytheshort-chainoligomers (Cp)6Cto

(Cp)12C is found (curve II), probably caused bya coopera-

tive actionofthese oligonucleotides. Howeverthe sharpin-

creaseoftemplateactivityatthe chain length13isindepen- dent oftheoligonucleotideconcentration.

(Cp)4(Up)S(Cp)1C oligonucleotides

In order to assay more "realistic" nucleotide sequences we

prepared oligonucleotides in which the two C-clusters ^are linkedbyaU-U-U-U-U-sequence.IntheexperimentofFig.

3 the template activity of the oligonucleotides

(Cp)4(Up)5(Cp).C

wasdeterminedas afunction ofn. Begin- ningwithⁿ ⁼4,correspondingtoanoverallchain length of

CHAINLENGTH

FIG. 2. Template activity of (Cp)0C oligomersas afunction of chain length. The incubation mixture (50 Ml) contained 50 mM Tris-HCl (pH 7.5), 10% (v/v) glycerol, 0.1 mM dithiothreitol, 10 mMMgC12,0.05 mM[y-32P]GTP(specificactivity500Ci/mol),4 ,gofQ6 replicase, and 2.5,M (Cp),C (curveI) or25 MM(Cp).C (curve II),asindicated.Incubationwasat300 for 5 min. A15iul al- iquotwas then appliedto DEAE-cellulose ^paper (Whatman DE 81). Theoligonucleotide productwasseparatedfrom ['y-32P]GTP and32PPibyelectrophoresisin 7%(v/v)formicacid for 10 hr at15 V/cm. The oligonucleotide products were cut out and their ra-

dioactivitiesweremeasuredby liquidscintillationcounting.

14,these model compounds direct theincorporationofAMP andGMP with steeplyincreasingefficiency. Thefollowing evidence^canbe offered for the actualsynthesisof the^com- plementary oligonucleotide ppp(Gp)8(Ap)5(Gp)3G when (Cp)4(Up)5(Cp)7C is used astemplate: (a) omission ofGTP from the incubation mixturecompletely suppressed the incorporation ofAMP. (b) Ina double labelexperimentusing [3H]ATP and [a-32P]GTP the molar ratio of incorporation

was found to be 1 AMP:1.9 GMP. (c) A nearest neighbor analysiswith [a-32PJGTP yieldsadinucleotide frequency of 92%GpGand 8%ApG.

(Aph(Cp)j4_jSC and(Up)(Cp)i2i4Coligonucleotides Theexperimentsdescribedsofar elucidatedthe minimalre-

quirementsfor template activity. Wenow canaskwhether

E 30000

0

z 0.

L 10000-

C4-u5 C4-U5-Ci C4-u5-C3 C4-U5-C5 C4-U5-C7 CHAINLENGTH

FIG. 3. Template activityof(Cp)4(Up)5(Cp).Coligomersasa

function of chainlength.The incubation mixture(50 Ml)contained 50 mMTris-HCl(pH 7.5), 10%glycerol,0.1 mMdithiothreitol,10 mMMgCl2,0.05 mMGTP,0.05 mM[a-32P]ATP (specific activity 500Ci/mol),4jugofQf replicase,and 10AMoligonucleotide,asindicated. Incubationwasat300 for5 min.Incorporatedradioactivi- tywasmeasuredasdescribedinFig.2.

-o-

POLY C

Biochemistry: Kfippers

and

Sumper

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2642 Biochemistry: Kuippers and Sumper

Table 2. Template activityofpolynucleotides pmolNMP in- pmolNMP in-

corporated(com- corporated(incuba- Poly- plete incubation tionmixture with-

nucleotide mixture) outGTP)

Poly(C) 4500(GMP) -

Poly(A) <5(UMP) -

(Ap)m(Cp)nC 3800(UMP) < 5(UMP)

Poly(U) <5(AMP) -

(Up)m(Cp)nC 1200(AMP) <5(AMP)

The incubation mixture (100 Ml) contained50mMTris-HCl (pH 7.5), 10%glycerol, 0.1 mMdithiothreitol, 10mMMgCl2, 0.2mM nucleosidetriphosphates(GTPand UTPorGTP andATP),oneof whichwaslabeled with14C(specific activity5Ci/mol),2.7zgof

Qf3 replicase, and 1 AM polynucleotide, asindicated. Incubation

wasat300for 10min.IncorporationwasmeasuredbytheMillipore

filtertechnique.

FIG. 4. Model oftemplate recognitionofphageRNAreplicas- es.(A) templateswithfixedtertiarystructure;(B)randomcopolymers;(C) 3'-terminalsequence ofMS2(-)strand(20).For details

seeDiscussion.

QB replicase is abletoread throughthe initiation sequence intoany nucleotide sequence. As model templates we chose

(Ap)7(Cp)4l-6C

and (Up)7(Cp)12-14C. Table 1 shows that theseoligonucleotides

efficiently

direct the incorporationof UMPand AMP, respectively. Whenaccount is taken ofthe different base compositions our model

templates (Table

1)

*turnedout tobetemplatesaseffectiveaspoly(C).

(Ap),(Cp)nC

and

(Up).(Cp)1C

polynucleotides

Although poly(A) and poly(U) are completely inactive as templatesforQB-replicase,thesepolymers became excellent templates after being linked with an initiation sequence at their 3'-terminus (Table 2). These experiments suggest that any polynucleotide linked to an initiation sequence can serveastemplatefor

Qo-replicase.

DISCUSSION

Our experiments with oligonucleotides demonstrate quite clearlythata C-clusteratthe 3'-endalong withasecond C- clusteradefined distance from the3'-terminustriggersiniti- ation ofRNA synthesis byQB replicase. A-lltemplate RNAs sequenced sofar(Fig. 1)fulfill this minimal requirementin their 3'-end regions. The only exception is QB (+) strand

Table 1. Templateactivityofsynthetic oligonucleotides

Oligonucleotide pmol NMPincorporated

(Cp),7C

550(GMP)

(Ap)6A <1 (UMP)

(Ap)7(Cp)14

,6C 395(UMP)

(Up)6u <1 (AMP)

(Up)7(Cp)

21,4C

195 (AMP)

(CP)4(Up)5(Cp)7C

205 (AMP)

The incubation mixture (100 gl) contained 50mM Tris-HCl (pH 7.5), 10%glycerol,0.1mMdithiothreitol, 10mMMgCl2,0.05mM nucleosidetriphosphates(GTPand UTPorGTP andATP),oneof which waslabeled with14C (specific activity 50Ci/mol), 4,g of Q, replicase, and10IMMoligonucleotide, asindicated. Incubation was at 300 for 10 min. Incorporated radioactivitywas determined asdescribedinFig.2.

RNA.

Remarkably,

thisRNAcannotbereplicated byQBre-

plicase

alone. The presence of at least one further protein factor

(6)

is necessary for

template

activity. Nucleotide ^se- quencesof

QB (+)

RNA

fragments

recoveredafter nuclease treatment from thereplicase binding complexwith Q,3

(+)

RNAarenotcommon tothe othertemplateRNAs. Itfollows that these

binding

sequencesareinvolvedinother

biological

functions

[e.g.,

repressoractionofQfl replicase (18)]andare not necessary to fulfill the minimal requirements for tem-

plate

activity.

Theproposedmodel oftemplaterecognition isabletoex-

plain

aparadoxicalpropertyofQBreplicase, namely, thatit is extremely specificagainstnaturally occurring RNAs and yet acceptsC-containing randomcopolymers. Only thosese- quencesable toofferthetwoC-clustersinthecorrect steric position can act as templates. Since naturally occurring RNAshavein

general

afixed tertiary structurethismecha- nism

efficiently

discriminates between templates and non-

templates. Onthe otherhand, RNA sequenceswithlittleor no tertiary structure, allowing more flexibility, can nearly

always

fulfill theinitiationconditions, iftheyhaveaC-clus-

ter at the3'-end and asecond C-cluster somewherefurther

in.

Recently it was shown that Qfl replicase generates de novo an apparently unlimited variety of

self-replicating-

RNAstructures(19). Sincenolongandcomplicated nucleotide sequences are necessary for template recognition, a

large number of RNAs can indeed fulfill the minimal re- quirementsandcanserve asactivetemplates.

Amodelillustratingthesepoints isshowninFig.4.

Furthermore, our model can be modified to explain the specificity of phage MS2, f2, or R17 replicases as well. In analogy to QB replicase these replicases accept poly(C) as active template (21) and all cognate RNAsalsocontaintwo C-C-C-clusters in a defined steric position. As can be seen

from the known3'-terminusof MS2(-)-strand (Fig. 4), the stericpositionof the internalC-C-C-cluster^isdifferent from that of

Qf,-active

templates. This displacement couldcause thelack ofcross-activityofreplicases andRNAtemplates of group I phages (QB) and group III phages (MS2, R17, f2, etc.).

It should be possibleto convert any desired RNA into a

template for

Qf3

replicase in a way analogous to that described forpoly(U) andpoly(A). Inprinciple, itshouldeven

bepossibletomodifyany RNAtobecomea

self-replicating

species.

Proc. Nat.Acad.Sci. USA72

(1975)

(4)

Proc. Nat.Acad.Sci. USA 72 (1975) 2643

We would like to thank Prof. M. Eigen for his encouragement andsupport of this work. We are also indebtedtoDr. Biebricher for many discussions and to Dr. Whooley forcorrecting our English.

Theexcellent technicalassistanceof R. Luce isgratefullyacknowl- edged.

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Biochemistry: Kfippers

and