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00925njm 22002777a 4500 dc Toribio, René author Díaz-López, Irene author Boskovic, Jasminka author Ventoso, Ivan author 2018-05-04 The topology and dynamics of the scanning ribosomal 43S pre-initiation complex (PIC) bound to mRNA and initiation factors (eIFs) are probably the least understood aspects of translation initiation in eukaryotes. Recently, we described a trapping mechanism in alphavirus that stalls the PIC during scanning of viral mRNA. Using this model, we were able to snapshot for the first time the eIF4A helicase bound to mRNA in a 48S initiation complex assembled in vitro. This interaction was only detected in the presence of the natural stem loop structure (DLP) located downstream from the AUG in viral mRNA that promoted stalling of the PIC, suggesting that DLP stability was enough to jam the helicase activity of eIF4A in a fraction of assembled 48S complexes. However, a substantial proportion of DLP mRNA molecules were effectively unwound by eIF4A in vitro, an activity that alphaviruses counteract in infected cells by excluding eIF4A from viral factories. Our data indicated that eIF4A-mRNA contact occurred in (or near) the ES6S region of the 40S subunit, suggesting that incoming mRNA sequences penetrate through the ES6S region during the scanning process. We propose a topological model of the scanning PIC and how some viruses have exploited this topology to translate their mRNAs with fewer eIF requirements. Nucleic Acids Res. 2018; 46 (8): 4176-4187. 10.1093/nar/gky071 1362-4962 0305-1048 Nucleic acids research 29415133 http://hdl.handle.net/20.500.12105/6503 43S PREINITIATION COMPLEX SECONDARY STRUCTURE HELICASE ACTIVITY CODON SELECTION FACTOR 4A FACTOR 4G EIF4A MECHANISM RIBOSOME INHIBITION Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex