Efficient translation initiation and optimal stability of most eukaryotic messenger RNAs depends on the formation of a closed-loop structure and the resulting synergistic interplay between the 5′ m7G cap and the 3′ poly(A) tail1,2. Evidence of eIF4G and Pab1 interaction supports the notion of a closed-loop mRNP3, but the mechanistic events that lead to its formation and maintenance are still unknown. Here we use toeprinting and polysome profiling assays to delineate ribosome positioning at initiator AUG codons and ribosome–mRNA association, respectively, and find that two distinct stable (resistant to cap analogue) closed-loop structures are formed during initiation in yeast cell-free extracts. The integrity of both forms requires the mRNA cap and poly(A) tail, as well as eIF4E, eIF4G, Pab1 and eIF3, and is dependent on the length of both the mRNA and the poly(A) tail. Formation of the first structure requires the 48S ribosomal complex, whereas the second requires an 80S ribosome and the termination factors eRF3/Sup35 and eRF1/Sup45. The involvement of the termination factors is independent of a termination event.
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We thank S. Kervestin for providing us with recombinant Pab1; J. McCarthy for eIF4E antibodies; A. Hinnebusch for the cdc33 strain; D. Bedwell for the plasmid-borne sup35-R419G allele; and members of the Jacobson laboratory for comments and discussions. This work was supported by a grant to A.J. from the National Institutes of Health.
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Amrani, N., Ghosh, S., Mangus, D. et al. Translation factors promote the formation of two states of the closed-loop mRNP. Nature 453, 1276–1280 (2008). https://doi.org/10.1038/nature06974
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