Proc. Nat. Acad. Sci. USAVol. 71, No. 2, pp. 436-440, February 1974

Two Eukaryotic Initiation Factors (IF-I and IF-II) of Protein Synthesis thatAre Required to Form an Initiation Complex with Rabbit Reticulocyte Ribosomes

(complex between two initiation factors/messenger ribonucleoproteins/synthetic oligoribonucleotides/puromycin)

LINDA M. CASHION AND WENDELL M. STANLEY, JR.

Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California, Irvine, Calif. 92664

Communicated by R. W. Gerard, September 27, 1973

ABSTRACT The formation of an initiation complexwith rabbit reticulocyte ribosomes and certain templatesis dependent upon the action of two initiation factors(IF-I and IF-II) of protein synthesis isolated from 0.5 MKCl extracts of rabbit reticulocyte polysomes. IF-I medi-ates the GTP-dependent, but template-independent,binding of the cytoplasmic initiator transfer RNA (rabbitliver) to a 40S ribosomal subunit. In the presence of a 60Sribosomal subunit and of AUG, AUG(U)25, or rabbit-globinmessenger ribonucleoproteins (but not of rabbit-globinmessenger RNAs), IF-II causes the transfer of the initi-ator transfer RNA to an initiation complex containing an80S ribosome. Although 5'-guanylylmethylenediphos-phonate can replace GTP as the cofactor used in bindingthe initiator transfer RNA to IF-I or to a 40S ribosomalsubunit, the subsequent transfer of the initiator transferRNA to an initiation complex requires GTP and cannottake place in the presence of 5'-guanylylmethylenedi-phosphonate. After the formation of this initiation com-plex, the initiator transfer RNA is bound in the P site ofthe 80S ribosome since exposure of the complex to puro-mycin releases the methionine residue from the initiatortransfer RNA as methionyl-puromycin.

IF-I and IF-II are dissociated from polysomes by 0.5 MKCI as a complex having a molecular weight of about370,000, and they remain in this form during and aftergel filtration. However, the two factors can be separatedby appropriate step elutions from DEAE-cellulose. Afterseparation of the two activities, the molecular weights ofIF-I and IF-II are about 150,000 and 220,000, respectively.

The major components of an initiation complex, involved ineukaryotic cytoplasmic protein biosynthesis, have beenidentified and include the 40S and 60S ribosomal subunits,the initiator tRNA (Met-tRNAMe), and an mRNA con-taining an appropriate initiator AUG codon (1). However, theidentities, physical properties, and functions of individualinitiation factors that control the formation of this initiationcomplex have not been well characterized. Previous reports(2-5) from our laboratory have described one initiation factor(IF-I), isolated from 0.5M KCl extracts of rabbit reticulocytepolysomes, which is required for the template-independentbinding of Met-tRNA Met (rabbit liver) to a 40S ribosomal

Met

Abbreviations: Met-tRNA, , methionyl-tRNA that can beenzymatically formylated (except for Met-tRNA Met from plants)by Escherichia coli enzymes and that functions as the initiatortRNA in the cytoplasm of eukaryotes; GDPCP, 5'-guanylyl-methylenediphosphonate; mRNPs, globin mRNAs with asso-ciated proteins that are released from rabbit reticulocyte poly-somes by treatment with EDTA; P site, peptidyl-tRNA bindingsite on the ribosome; A site, aminoacyl-tRNA binding site onthe ribosome; IF, initiation factor.

subunit (rabbit reticulocytes). In the absence of ribosomalsubunits, IF-I forms a stable ternary complex (2, 4-8) withGTP [or 5'-guanylylmethylenediphosphonate (GDPCP)(2)] and with the cytoplasmic initiator tRNA from alleukaryotic sources tested (rabbit reticulocytes, rabbit liver,chicken liver, and rat liver) (4); these ternary complexes areretained by Millipore filters (2, 4-8). Ternary complexformation is blocked by ammonium aurintricarboxylate (3) orby prior reaction of IF-I with any one of the sulfhydryl re-agents tested [N-ethylmaleimide, cystamine, and 5,5'-dithio-bis-(2-nitrobenzoate)] (4). IF-I has been purified from avariety of eukaryotic tissues and cells (rabbit reticulocytes,rabbit liver, chicken reticulocytes, chicken liver, and chickembryonic leg muscle), and no differences were found betweenthese IF-I preparations with respect to either their biologicalactivities or their physical properties (4). The molecularweights of all IF-I activities in 0.5 M KCl polysomal extractswere determined to be approximately 370,000 (4).We now report the detection, purification, and function of a

second eukaryotic initiation factor (IF-II) of protein syn-thesis isolated from rabbit reticulocyte IF-I of 370,000molecular weight. The IF-I and IF-II activities in the com-plex of 370,000 molecular weight were separated by sequentialstep elutions from DEAE-cellulose. IF-II is required for thetransfer of Met-tRNAm" (rabbit liver) from a 40S ribosomalsubunit to an initiation complex in the presence of 60Sribosomal subunits. This transfer is stimulated greatly bythe initiator AUG triplet, by the defined oligoribonucleotideAUG(U)25, and by rabbit-globin messenger ribonucleoproteins(mRNPs), but not by rabbit-globin mRNAs. After the IF-II-mediated transfer of the initiator tRNA, the tRNA is boundto the P site of the 80S ribosome where the methionine residueis available for reaction with puromycin. All functions of IF-IIrequire GTP in an enzymatically hydrolyzable form.

MATERIALS AND METHODS

The preparations of high-specific-activity i_[35S]methionine(9), [35S]Met-tRNAmet (rabbit liver) (10, 11), and template-and factor-free rabbit reticulocyte ribosomes (12) have beendescribed. Rabbit-globin mRNPs were isolated from polysomesby treatment with EDTA followed by zone velocity sedimen-tation through preformed sucrose density gradients (13), andthen were recovered by high-speed centrifugation (5). GlobinmRNAs were prepared by phenol deproteinization of the globinmRNPs. Assays for IF-I activity by Millipore filter reten-tion of the IF-I 135S]Met-tRNAMet GTP ternary complex(2, 5) and by IF-I-dependent [35S]Met-tRNAmet binding

436

Two Eukaryotic Initiation Factors 437

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FIG. 1. Steps leading to the formation of an initiation com-plex. Binding reactions were prepared as described in Methods.As specified below, 825 pmol of AUG or 680 pmol of AUG(U)25were added before the second incubation. Reactions were layeredon 10-25% (w/v) exponential sucrose gradients made in bufferB. The gradients were centrifuged at 2 for 6 hr at 32,000 rpmin a Spinco type SW 40 Ti rotor. Additions are as follows: (A)IF-I; (B) IF-I + AUG; (C) IF-I + AUG(U)25; (D) IF-I +IF-II; (E) IF-I + IF-II + AUG; (F) IF-I + IF-II + AUG(U)25.

to the 40S ribosomal subunit (4, 5) have been reported. Anassay for IF-II activity by reaction of [35S]Met-tRNAm'tin an initiation complex with puromycin also has been de-scribed (5). GDPCP and puromycin were from Miles Labora-tories, Inc. and Nutritional Biochemicals, Inc., respectively.

Purification of the Initiation Factors. IF-I and IF-II arecopurified as the complex of 370,000 molecular weight, essen-tially as described (4, 5), by gel filtration. The two initiationfactors then are separated by DEAE-cellulose chromatog-raphy (5). A 0.5M KCl extract of rabbit reticulocyte polysomesis applied to a column of Sephadex G-200 (Pharmacia FineChemicals, Inc.), equilibrated and developed in the cold (40)with 50 mM KCl in buffer A [25mM potassium phosphate (pH7.0)-i mM 2-mercaptoethanol-10% (v/v) glycerol]. Thecomplex of IF-I and IF-II elutes immediately after the ex-cluded volume of the column. The eluate, containing bothIF-I and IF-II activities, is applied to a small column ofDEAE-cellulose (Whatman DE-52, 1 mequiv/g of dryweight) operated in the cold. IF-I is eluted with 0.125 M KClin buffer A; the column then is washed with 0.15 M KCl inbuffer A and this wash is discarded. IF-II is eluted with 0.25M KCl in buffer A. The two solutions containing the separatedinitiation factors are stored at -70 and are stable for severalmonths.

IF-I- and IF-II-Mediated Binding of [9S]Met-tRNAiet toRibosomal Subunits and to Ribosomes (5). An IF-I ternarycomplex is generated in situ by a preliminary incubation of 11jsg of IF-I with 1.5 pmol of [35S]Met-tRNA Met and 70 nmolof GTP (or of GDPCP) as described (4, 5). An initiationcomplex then is allowed to form in a second incubation at 250for 10 min in 0.1 ml of buffer B [75mM KCl-50 mM Tris - HCl

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ELUTION VOLUME (ML)FIG. 4. Molecular-weight determinations of IF-I and of IF-II.

(A) Logarithmic plot of molecular weight against elution volume.1, Urease; 2, complex of IF-I and IF-II; 3, catalase; 4, IF-IIseparated from IF-I; 5, aldolase; 6, IF-I separated from IF-II.(B) Relative activities of initiation factors in the column eluate.0, IF-I, in a complex with IF-II; a relative value of 100 = 37pmol of ["S]Met-tRNAmet bound per ml. *, IF-II, separatedfrom IF-I; 100 = 7.1 pmol of [a*S]methionyl-puromycin formedper ml. A, IF-I, separated from IF-II; 100 = 1.9 pmol of [3ES]Met-tRNAmet bound per ml. All assays used are described in Methods.

(14). The sample is centrifuged briefly to separate the phases.[At pH 8.0, the extraction efficiency is 50% (14).] One mil-liliter of the ethyl acetate phase is removed, mixed with 4 mlof Bray's solution (15), and counted in a liquid scintillationspectrometer.

Molecular Weight Determinations. A 0.9 X 114-cm columnof Sepharo