Cotranslational Folding Stimulates Programmed Ribosomal Frameshifting in the Alphavirus Structural Polyprotein

Haley R. Harrington; Matthew H. Zimmer; Laura M. Chamness; Veronica Nash; Wesley D. Penn; Thomas F. Miller; Suchetana Mukhopadhyay; Jonathan P. Schlebach

Coarse-grained molecular dynamics (CGMD) simulations are based on a previously developed and tested approach. 44 The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/790444 doi: bioRxiv preprint necessary to describe the system are available in previous published work. 45 Integration is performed using overdamped Langevin dynamics with a diffusion coefficient of 253 nm 2 /s and a time step of 300 ns. Despite the significant simplifications involved in this model, the CGMD model has proven accurate in capturing the integration probabilities, topology distributions, and forces experienced in previous studies. 44, 45, 47 In order to obtain the distribution of topologies for various polyprotein mutants, the translation and integration of each sequence was simulated 560 times. In order to reduce computational cost, simulations only included the first three TMDs of the alphavirus polyprotein. In order to focus on the topological preferences of TM2, restraints were applied to enforce that TM1 adopts its native topological orientation. Pulling force measurements were performed by pausing translation when the -1PRF site resides within the ribosomal peptidyl transfer center. During this pause, the final bead was fixed in place and the force on the bead exerted by the nascent chain was measured along the translocon channel axis. Due to the truncation of the exit tunnel in our model, the final bead corresponds to the amino acids 27 residues N-terminal of the -1PRF site. Translation is paused for 3 seconds, which is equivalent to the time it would take to translate 5 beads. This relatively short time window ensures that the distribution of polyprotein topologies is not affected by the pause.