The nature of the coupling between the stalling of the elongated nascent peptide chain in the ribosome and its insertion through the translocon is analyzed, focusing on the recently discovered biphasic force that overcomes the stalling barrier. The origin of this long-range coupling is explored by coarse-grained simulations that combine the translocon (TR) insertion profile and the effective chemical barrier for the extension of the nascent chain in the ribosome. Our simulation determined that the inserted H segment is unlikely to climb the TR barrier in parallel with the peptide synthesis chemical step and that the nascent chain should first overcome the chemical barriers and move into the ribosome-TR gap region before the insertion into the TR tunnel. Furthermore, the simulations indicate that the coupled TR-chemistry free energy profile accounts for the biphasic force. Apparently, although the overall elongation/insertion process can be depicted as a tug-ofwar between the forces of the TR and the ribosome, it is actually a reflection of the combined free-energy landscape. Most importantly, the present study helps to relate the experimental observation of the biphasic force to crucial information about the elusive path and barriers of the TR insertion process.he elongation of nascent peptide chains during their synthesis by the ribosome and the translocon (TR)-assisted insertion of the generated chain into the membrane are coupled in an intriguing way. A glimpse into this coupling has been provided recently by the von Heine group (1), who observed an interplay between the stalling of the elongation process and the TR insertion process. More specifically, it was found that in cases when the elongation stalls due to the presence of an RXXP-type sequence, the process is reactivated by the force generated in the TR for different lengths of the inserted chain. While this finding is very exciting, it is not clear what the exact origin of the applied force is and how it can be coupled to the elongation process, where the insertion into the TR involves an uphill penetration process.The peptide elongation process and the subsequent insertion are described schematically in Fig. 1 for the system studied by von Heijne and coworkers (1). As seen from the figure, after the peptide bond is synthesized, the extended peptide moves through the TR and then inserts into the membrane. However, the chain extension process can be stalled during the elongation process of the RXXP and other sequences (2-6). This stall reflects, most probably, the increase in the activation barrier for the peptide bond formation, due in part to changes in the preorganization of the active site (SI Text). As demonstrated in ref. 1, for some lengths of the linker (denoted by L) the stalling is released as the H segment (a 19-residue leucine-alanine-based peptide, which was introduced into the leader peptidase protein) passes through the TR (Fig. 1 B and C). On the other hand, for other lengths the stalling cannot be overcome and the ribosome remains atta...