Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arise due the poor sensitivity, low time resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes and allows for continuous acquisition with a time resolution in the order of seconds, yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigor unattainable through classical methods.

Abbreviations:AA -antimycin A; IMM -inner mitochondrial membrane; mt-11S -mitochondrial-targeted 11S; H6 11S -Nterminal 6xHis tagged version of 11S; OMM -outer mitochondrial membrane; OVA -oligomycin, valinomycin and antimycin A (full depolarisation cocktail); MTS -mitochondrial targeting sequence, NanoBiT -NanoLuc Binary Technology; PAM -presequence-translocase-associated import-motor; pep86high-affinity version of NanoBiT small fragment; pep114 -small fragment of NanoBiT; PMF -proton motive force; TIM -translocase of the inner membrane; TOM -translocase of the outer membrane; 11Slarge fragment of the NanoBiT; Δ Ψ -membrane potential. 2 ABSTRACTProtein translocation is a fundamental process in biology. Major gaps in our understanding of this process arises due the poor sensitivity, low time-resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes, and allows continuous acquisition with a time-resolution in the order of seconds -yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system, and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigour unattainable through classical methods.

a b s t r a c tPicornavirus RNAs initiate translation using a 5 0 end-independent mechanism based on internal ribosome entry site (IRES) elements. Despite performing similar functions, IRES elements present in genetically distant RNAs differ in primary sequence, RNA secondary structure and trans-acting factors requirement. The lack of conserved features amongst IRESs represents obstacles for the understanding of the internal initiation process. RNA structure is tightly linked to picornavirus IRES activity, consistent with the conservation of RNA motifs. This study extends the functional relevance of evolutionary conserved motifs of foot-and-mouth disease virus (FMDV) IRES. SHAPE structural analysis of mutant IRESs revealed local changes in RNA flexibility indicating the existence of an interactive structure constrained by lateral bulges that maintain the RNA conformation necessary for IRES-mediated translation.