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We have developed a new technique^ called 'toeprinting/ which has allowed a study of the tRNA-binding properties of Escherichia coli translation initiation complexes. In response to natural mRNAs, the initiator tRNA and a variety of elongator tRNAs bind to the same tRNA-binding site on the 308 ribosomal subunit as long as a cognate codon is present near the Shine and Dalgarno sequence. The selection of the initiator tRNA in 30S initiation complexes is accomplished by initiation factors IF2 and IF3. 70S ribosomes accept both initiator tRNA and elongator tRNAs on natural mRNAs, much like 30S ribosomal subunits; IF3 and IF2 do not, however, select the initiator tRNA on 70S initiation complexes unless the initiation factor IFl is present.

Lysis gene S of phage lambda has a 107 codon reading frame beginning with the codons Met1‐Lys2‐Met3. Genetic data have suggested that translational initiation occurs at both Met1 and Met3, generating two polypeptides, S107 and S105 respectively. We have proposed a model in which the proper scheduling of lysis depends on the partition of translational initiations between the two start codons. Here, using in vitro methods, we show that two stem‐loop structures, one immediately upstream of the reading frame and a second approximately 10 codons within the gene, control the partitioning event. Utilizing primer‐extension inhibition or ‘toeprinting’, we show that the two S start codons are served by two adjacent Shine‐Dalgarno sequences. Moreover, the timing of lysis supported by the wild‐type and a number of mutant alleles in vivo can be correlated with the ratio of ternary complex formation over Met1 and Met3 in vitro. Thus the regulation of the S gene is unique in that the products of two adjacent in‐frame initiation events have opposing function.

Initiation factors are used by Escherichia cob to select the initiator tRNA over elongator tRNAs during translation initiation. IF3 appears to "inspect" the anticodon end of the tRNA, probably along with the initiation codon. The anticodon stem and loop of the initiator tRNA, together with part of the initiation codon of the mRNA, can be thought of as a unit. Changes made in the anticodon stem, the anticodon loop, or the anticodon of an initiator tRNA fragment result in a loss of selection by IF3 in an in vitro assay for translation initiation. IF3 allows the selection of an initiator tRNA anticodon stem and loop fragment on GUG and UUG codons but does not select that tRNA fragment in response to AUU. Escherichia coli initiation factor IF3 is involved in several aspects of translation initiation; IF3 (together with IF1) helps in the dissociation of 70S ribosomes into 30S and 50S subunits (Kaempfer 1972;Godefroy-Colburn et al. 1975), catalyzes the formation of 30S initiation complexes (Wintermeyer and Gualerzi 1983), and selects the initiator tRNA against elongator tRNAs in those complexes . The selection of the initiator tRNA by IF3 is based on the unique RNA part of the initiator tRNA (Risuleo et al. 1976), and, most likely, sequences located in the anticodon stem and loop . The three GC base pairs of the anticodon stem adjacent to the loop of the initiator tRNA are important for initiation complex formation and in vitro translation (Seong and RajBhandary 1987), suggesting that this part of the anticodon stem might be important for recognition by IF3. Berkhout et al. (1986) showed that IF3 excludes initiator tRNA on a UUU codon, as though the initiation codon, itself, might be part of the recognition motif explored by IF3 during selection of the initiator tRNA.We have examined those features of the initiator tRNA that are required for selection by IF3. Using toeprinting (Winter et al. 1987;Hartz et al. 1988Hartz et al. , 1989McPheeters et al. 1988;Blasi et al. 1989;Schaefer et al. 1989) as the assay for tRNA binding in 30S initiation complexes, we tested initiator tRNA fragments and several mutant fragments for their ability to be selected by IF3 on various initiation codons. ResultsTo analyze 30S complex formation on various translation initiation sites, we used the extension inhibition technique, also called toeprinting, cDNA synthesis on a template mRNA is terminated when the reverse transcriptase encounters a 30S ribosomal subunit plus tRNA bound on the mRNA. The short cDNA is visualized as a toeprint band on a sequencing gel. The toeprint usually appears 15 bases downstream of the A of the initiation codon if initiator tRNA is bound in the complex or at + 15 from the first base of the cognate codon if elongator tRNA is bound. When compared to the band of the fulllength cDNA, the toeprint band is a quantitative measure of ternary complex formation . Variations of A22~ etUsing the toeprinting assay, we have shown previously that the synthetic initiator tRNA fragment A22~ et (Fig. 1) behaves very similarly ...

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