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Two initiation factors, 1 (IF1) and 3 (IF3), contribute to quality control for formation of 30S preinitiation complex (30S PIC), while the GTPase IF2 facilitates stable initiator tRNA binding and promotes subunit association. However, the molecular mechanism of IF1 action and the regulatory role of IF2-mediated GTP hydrolysis and inorganic phosphate (Pi) release remain poorly understood. Using ensemble cryo-EM integrated with fast-kinetics, we delineate the translation initiation pathway involving IF1 and IF2. We show that IF1 transiently associates with the 30S subunit and interferes with the formation of multiple inter-subunit bridges. IF2 promotes subunit association by stabilizing the 30S PIC through interactions mediated by its N-terminal domains. IF1 departure happens after or concomitant with GTP hydrolysis, following which the inter-subunit bridges establish. Then Pi release triggers remodeling of IF2 followed by its departure from the 70S initiation complex. These findings reveal how the coordinated interplay of IF1 and IF2 with the ribosome ensures translational fidelity and plays crucial role for formation of elongation-competent 70S.<\/jats:p>","DOI":"10.64898\/2026.06.26.734871","type":"posted-content","created":{"date-parts":[[2026,6,29]],"date-time":"2026-06-29T14:55:11Z","timestamp":1782744911000},"source":"Crossref","is-referenced-by-count":1,"title":["Molecular mechanism of IF1- and IF2-driven translation initiation in bacteria"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5277-5938","authenticated-orcid":false,"given":"Gabriel Soares","family":"Guerra","sequence":"first","affiliation":[{"name":"Central European Institute of Technology, Masaryk University, Brno, Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hassan","family":"Zafar","sequence":"additional","affiliation":[{"name":"Central European Institute of Technology, Masaryk University, Brno, Czech 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