Nature © Macmillan Publishers Ltd 19988Eukaryotic ribosomes requireinitiation factors 1 and 1Ato locate initiation codonsTatyana V. Pestova*†, Sergei I. Borukhov* & Christopher U. T. Hellen** Department of Microbiology and Immunology, State University of New York Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, New York 11203, USA†A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia........................................................................................................................................................................................................................................................The scanning model of translation initiation is a coherent description of how eukaryotic ribosomes reach the initiationcodon after being recruited to the capped 59 end of messenger RNA. Five eukaryotic initiation factors (eIF 2, 3, 4A, 4Band 4F) with established functions have been assumed to be sufficient to mediate this process. Here we report thateIF1 and eIF1A are also both essential for translation initiation. In their absence, 43S ribosomal preinitiationcomplexes incubated with ATP, eIF4A, eIF4B and eIF4F bind exclusively to the cap-proximal region but are unable toreach the initiation codon. Individually, eIF1A enhances formation of this cap-proximal complex, and eIF1 weaklypromotes formation of a 48S ribosomal complex at the initiation codon. These proteins act synergistically to mediateassembly of ribosomal initiation complexes at the initiation codon and dissociate aberrant complexes from the mRNA.The ribosomal scanning model describes the basic steps of trans-lation initiation on most eukaryotic mRNAs1,2. In this process, a 43Scomplex, consisting of a ribosomal 40S subunit, eIF3 and an eIF2–GTP–initiator tRNA complex, binds mRNA at its 59 end and scansdownstream until it locates the initiation codon. First, eIF4F bindsthe capped 59 end of the mRNA and, with eIF4A and eIF4B, createsan unstructured cap-proximal binding site for the 43S complex.This complex scans to the first downstream AUG triplet, which actsas the initiation codon. eIF5 stimulates GTP hydrolysis and releaseof factors from the resulting 48S complex, leaving the initiator tRNAin the P-site of the 40S subunit. The ribosomal 60S subunit thenjoins the 40S subunit and protein synthesis begins. Other factors,including eIF1 and eIF1A, have been implicated in the initiation oftranslation but their function remains obscure3.Ribosomal binding to the end of an mRNA does not position itat the initiation codon, which is usually 50–100 nucleotides away.The 43S complex is thought to scan downstream, searching for theinitiation codon. This model poses three basic questions. (1) Whichfactors are required for attachment of 43S complexes to cappedmRNAs? (2) How does the 43S complex move on the mRNA, andwhich factors are required for this process? (3) How do componentsof the 43S complex interact with and inspect mRNA duringscanning to recognize and reject mismatched interactions betweentriplets in the mRNA and the anticodon of initiator tRNA before thecorrect initiation codon is selected?We have developed methods to reconstitute initiation frompurified components and accurately to map the resulting initiationcomplexes on mRNAs4–6. Here we have reconsituted early stages ininitiation on natural capped b-globin mRNA and identified essen-tial, unanticipated activities of eIF1 and eIF1A. eIF1, eIF1A, eIF4A,eIF4B and eIF4F are sufficient for 43S complexes to bind cappedmRNAs and to form 48S complexes at the initiation codon. WheneIF1 and eIF1Awere omitted, 43S complexes bound near the 59 capbut did not reach the initiation codon. eIF1A enhanced the forma-tion of these 59-terminal complexes in the presence of the other fivefactors; in their presence, eIF1 slightly stimulated 48S complexformation and dissociation of aberrant 59 terminal complexes.These factors have distinct, synergistic activities that are requiredtogether for 48S complex assembly at the initiation codon.Ribosome recruitment of capped mRNARibosomal 48S complexes were assembled in vitro on b-globinmRNA using purified factors (Fig. 1a). The position of 40S subunitson the mRNA in these complexes as mapped by toeprinting, whichinvolves extension by reverse transcriptase of a primer annealed to atemplate RNA to which a ribosome is also bound. Synthesis ofcomplementary DNA is arrested by the bound complex, yielding atoeprint at its leading edge that can be located on a sequencing gel.48S complexes assembled on b-globin mRNA yield stops 15, 16 and17 nucleotides downstream of the initiation codon7.A ribosomal ‘complex I’, assembled from 40S subunits, initiatortRNA, eIF2, eIF3, eIF4A, eIF4B and eIF4F, yielded prominenttoeprints 21–24 nucleotides from the 59 end of the mRNA (Fig.2a, lane 3). Complex I did not form if 40S subunits, initiator tRNA,eIF2, eIF3 or eIF4F individually, or eIF4A, eIF4B and eIF4F together,were omitted, or if ATP was substituted by AMP–PNP (Fig. 2a,lanes 1–3, 2b, lanes 1–7, 10). The formation of complex I wasgreatly increased by eIF4B (Fig. 2b, lane 8). 43S complexes andeIF4A, eIF4B and eIF4F are therefore unable to form 48S complexes,and instead form ribosomal complexes near the 59 terminal cap.Parallel experiments using a-globin mRNA led to an identicalconclusion. Ribosomal complexes yielded toeprints 16 and 23nucleotides from the 59 end of this mRNA, but not at the initiationcodon (data not shown).Assembly of 48S complexes48S complexes assemble correctly in rabbit reticulocyte lysate(RRL)7, which we therefore used as a source from which to purifyadditional factor(s) required for assembly of the 48S complex. The0.5 M KCl ribosomal salt wash was divided into 0–40%, 40–50%and 50–70% ammonium sulphate precipitation fractions. The 50–70% fraction contained most of the activity that promoted assemblyof a 48S complex (complex II) at the initiation codon on addition toreactions that contained 43S complexes and eIF4A, eIF4B and eIF4F(Fig. 2a, lane 4). This fraction was separated by elution from DEAEcellulose into 0.1 M KCl and 0.25 M KCl fractions that together hadthe same activity as the starting material (Fig. 1b). The 0.25 M KClfraction doubled complex I formation but did not promote com-plex II formation; inclusion of the 0.1 M KCl fraction in similarreactions yielded small amounts of complex II without significantlyaltering