articles Eukaryotic ribosomes require initiation factors 1 and 1A to locate initiation codons Tatyana 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 initiation codon after being recruited to the capped 59 end of messenger RNA Five eukaryotic initiation factors eIF 2 3 4A 4B and 4F with established functions have been assumed to be sufficient to mediate this process Here we report that eIF1 and eIF1A are also both essential for translation initiation In their absence 43S ribosomal preinitiation complexes incubated with ATP eIF4A eIF4B and eIF4F bind exclusively to the cap proximal region but are unable to reach the initiation codon Individually eIF1A enhances formation of this cap proximal complex and eIF1 weakly promotes formation of a 48S ribosomal complex at the initiation codon These proteins act synergistically to mediate assembly of ribosomal initiation complexes at the initiation codon and dissociate aberrant complexes from the mRNA The ribosomal scanning model describes the basic steps of translation initiation on most eukaryotic mRNAs1 2 In this process a 43S complex consisting of a ribosomal 40S subunit eIF3 and an eIF2 GTP initiator tRNA complex binds mRNA at its 59 end and scans downstream until it locates the initiation codon First eIF4F binds the capped 59 end of the mRNA and with eIF4A and eIF4B creates an unstructured cap proximal binding site for the 43S complex This complex scans to the first downstream AUG triplet which acts as the initiation codon eIF5 stimulates GTP hydrolysis and release of factors from the resulting 48S complex leaving the initiator tRNA in the P site of the 40S subunit The ribosomal 60S subunit then joins the 40S subunit and protein synthesis begins Other factors including eIF1 and eIF1A have been implicated in the initiation of translation but their function remains obscure3 Ribosomal binding to the end of an mRNA does not position it at the initiation codon which is usually 50 100 nucleotides away The 43S complex is thought to scan downstream searching for the initiation codon This model poses three basic questions 1 Which factors are required for attachment of 43S complexes to capped mRNAs 2 How does the 43S complex move on the mRNA and which factors are required for this process 3 How do components of the 43S complex interact with and inspect mRNA during scanning to recognize and reject mismatched interactions between triplets in the mRNA and the anticodon of initiator tRNA before the correct initiation codon is selected We have developed methods to reconstitute initiation from purified components and accurately to map the resulting initiation complexes on mRNAs4 6 Here we have reconsituted early stages in initiation on natural capped b globin mRNA and identified essential unanticipated activities of eIF1 and eIF1A eIF1 eIF1A eIF4A eIF4B and eIF4F are sufficient for 43S complexes to bind capped mRNAs and to form 48S complexes at the initiation codon When eIF1 and eIF1A were omitted 43S complexes bound near the 59 cap but did not reach the initiation codon eIF1A enhanced the formation of these 59 terminal complexes in the presence of the other five factors in their presence eIF1 slightly stimulated 48S complex formation and dissociation of aberrant 59 terminal complexes These factors have distinct synergistic activities that are required together for 48S complex assembly at the initiation codon Ribosome recruitment of capped mRNA Ribosomal 48S complexes were assembled in vitro on b globin 854 mRNA using purified factors Fig 1a The position of 40S subunits on the mRNA in these complexes as mapped by toeprinting which involves extension by reverse transcriptase of a primer annealed to a template RNA to which a ribosome is also bound Synthesis of complementary DNA is arrested by the bound complex yielding a toeprint at its leading edge that can be located on a sequencing gel 48S complexes assembled on b globin mRNA yield stops 15 16 and 17 nucleotides downstream of the initiation codon7 A ribosomal complex I assembled from 40S subunits initiator tRNA eIF2 eIF3 eIF4A eIF4B and eIF4F yielded prominent toeprints 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 was greatly increased by eIF4B Fig 2b lane 8 43S complexes and eIF4A 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 identical conclusion Ribosomal complexes yielded toeprints 16 and 23 nucleotides from the 59 end of this mRNA but not at the initiation codon data not shown Assembly of 48S complexes 48S complexes assemble correctly in rabbit reticulocyte lysate RRL 7 which we therefore used as a source from which to purify additional factor s required for assembly of the 48S complex The 0 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 assembly of a 48S complex complex II at the initiation codon on addition to reactions that contained 43S complexes and eIF4A eIF4B and eIF4F Fig 2a lane 4 This fraction was separated by elution from DEAE cellulose into 0 1 M KCl and 0 25 M KCl fractions that together had the same activity as the starting material Fig 1b The 0 25 M KCl fraction doubled complex I formation but did not promote complex II formation inclusion of the 0 1 M KCl fraction in similar reactions yielded small amounts of complex II without significantly altering formation of complex I the main product data not shown The active constituents in these fractions were purified by chromatography Fig 1b and were assayed after mixing using toeprinting after each step to identify fractions and proteins that Nature Macmillan Publishers Ltd 1998 NATURE VOL 394 27 AUGUST 1998 8 articles promoted complex II formation Apparently homogenous proteins of relative