A method of analysis of translation initiation complexes by toeprinting has recently acquired a wide application to investigate molecular mechanisms of translation initiation in eukaryotes. So far, this very fruitful approach was used when researchers did not aim to discriminate between patterns of toeprints for 48S and 80S translation initiation complexes. Here, using cap-dependent and internal ribosomal entry site (IRES)-dependent mRNAs, we show that the toeprint patterns for 48S and 80S complexes are distinct whether the complexes are assembled in rabbit reticulocyte lysate or from fully puri¢ed individual components. This observation allowed us to demonstrate for the ¢rst time a delay in the conversion of the 48S complex into the 80S complex for L L-globin and encephalomyocarditis virus (EMCV) RNAs, and to assess the potential of some 80S antibiotics to block polypeptide elongation. Besides, additional selection of the authentic initiation codon among three consecutive AUGs that follow the EMCV IRES was revealed at steps subsequent to the location of the initiation codon by the 40S ribosomal subunit. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

The reconstitution of translation initiation complexes from purified components is a reliable approach to determine the complete set of essential canonical initiation factors and auxiliary proteins required for the 40S ribosomal subunit to locate the initiation codon on individual mRNAs. Until now, it has been successful mostly for formation of 48S translation initiation complexes with viral IRES elements. Among cap-dependent mRNAs, only globin mRNAs and transcripts with artificial 5 leaders were amenable to this assembly. Here, with modified conditions for the reconstitution, 48S complexes have been successfully assembled with the 5 UTR of beta-actin mRNA (84 nucleotides) and the tripartite leader of adenovirus RNAs (232 nucleotides), though the latter has been able to use only the scanning rather then the shunting model of translation initiation with canonical initiation factors. We show that initiation factor 4B is essential for mRNAs that have even a rather moderate base pairing within their 5 UTRs (with the cumulative stability of the secondary structure within the entire 5 UTR < ؊13 kcal/mol) and not essential for beta-globin mRNA. A recombinant eIF4B poorly substitutes for the native factor. The 5 UTRs with base-paired G residues reveal a very sharp dependence on the eIF4B concentration to form the 48S complex. The data suggest that even small variations in concentration or activity of eIF4B in mammalian cells may differentially affect the translation of different classes of capdependent cellular mRNAs.Translation initiation is now recognized to play an important role in gene expression in eukaryotes in the course of viral infection, nutritional adaptation, stress, apoptosis, development and cell division (11). However, in most cases, the molecular mechanisms for this translational control remain mysterious. This is largely accounted for by our poor understanding of the molecular events that underlie the basic mechanisms of translation initiation in eukaryotes, especially in mammals. As mammalian systems are poorly amenable to genetic approaches, alternative ways need to be elaborated to address this problem. To this end, it was suggested several years ago to use an approach that is based on the assembly of translation initiation complexes from individual translational components combined with toeprinting (22). (Toeprinting is based on the primer extension inhibition of reverse transcription from an oligodeoxynucleotide, which is hybridized 3Ј to the initiation codon of an mRNA. The arrest of reverse transcriptase always occurs at the same positions, ϩ16 to ϩ18 nucleotides 3Ј of A in the AUG initiation codon.) This approach has proven to be fruitful in the elucidation of the function of several viral internal ribosome entry sites (IRES) (11). Surprisingly, it has proven to be unsuccessful with capdependent transcripts if their 5Ј UTRs comprised even short stem-loop structures enriched in G-C pairs (26). Among mammalian mRNAs with natural 5Ј UTRs, only globin mRNAs have been reported to result in high yiel...

Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an mRNA was supplied with a 5 leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus.More than a decade ago, we showed using the toeprint assay that the prokaryotic leaderless mRNA coding for the cI repressor of lambda phage assisted by initiator Met-tRNA was able to bind directly to undissociated 70S ribosomes in the absence of initiation factor 1 (IF1), IF2, and IF3 (1). Under the same conditions, no initiation complex was observed with the 30S ribosomal subunit. In the presence of initiation factors, the 30S subunit invariably bound to internal sites on the mRNA immediately downstream of the closest Shine-Dalgarno (SD) sequence, whereas undissociated 70S ribosome strongly preferred the initiation triplet as close as possible to the 5Ј end of the mRNA. These studies were substantially extended and confirmed in other laboratories both in vitro and in vivo (12,13,15,28) and resulted in similar conclusions. Natural leaderless mRNAs have been identified in all kingdoms of life (see references 10 and 11). This class of mRNAs is well represented in archaea and mammalian mitochondria. Although they have not yet been found among cellular mammalian mRNAs, leaderless monocistronic N and dicistronic N-P RNAs of measles virus were found to be associated with polysomes in infected mammalian cells (4). In addition, it is unlikely that a systematic search for such mRNAs in mammalian cells has ever been undertaken. There is one well-documented example of a translatable leaderless mRNA in unicellular eukaryotes. It encodes a variant specific surface protein of Giardia lamblia and is certainly translated in these cells (14). Leaderless mRNAs are efficiently translated in mammalian cell-free systems (see reference 12). However, it is not known whether they use a similar (80S) pathway for translation initiation in eukaryotes, given the remarkable quantitative and qualitative differences in the organization of eukaryotic and prokaryotic t...