Identifying Strategies to Experimentally Probe Multidimensional Dynamics in the Ribosome

Abstract

The ribosome is a complex biomolecular machine that utilizes large-scale conformational rearrangements to synthesize proteins. For example, during the elongation cycle, the "head" domain of the ribosomal small subunit (SSU) is known to undergo transient rotation events that allow for movement of tRNA molecules (i.e., translocation). While the head may exhibit rigid-body-like properties, the precise relationship between experimentally accessible probes and multidimensional rotations has yet to be established. To address this gap, we perform molecular dynamics simulations of the translocation step of the elongation cycle in the ribosome, where the SSU head spontaneously undergoes rotation and tilt-like motions. With this data set (1250 simulated events), we used statistical and information-theorybased measures to identify possible single-molecule probes that can isolate SSU head rotation and head tilting. This analysis provides a molecular interpretation for previous single-molecule measurements, while establishing a framework for the design of nextgeneration experiments that may precisely probe the mechanistic and kinetic aspects of the ribosome.

The energy landscape of the ribosome

“…Despite this limitation, Nishima et al claimed that their tilt‐like model was inconsistent with the single‐molecule observations, and therefore tilting is generally incompatible with experimental measurements. Interestingly, when the simulated trajectories of Nguyen and Whitford were re‐analyzed and compared with the experimental measurements, [ 150 ] the predicted tilting motion was shown to be associated with an increase (relative to INT2) in L5‐S13 distance of approximately 5 Å (Figure 3g), which is similar to the change expected based on the FRET measurements. This is in stark contrast to the Nishima tilting model, in which there is a reported 10.1 Å decrease in L5‐S13 distance, indicating the Nishima and Nguyen tilting models are fundamentally different.…”

“…Despite this limitation, Nishima et al claimed that their tilt-like model was inconsistent with the single-molecule observations, and therefore tilting is generally incompatible with experimental measurements. Interestingly, when the simulated trajectories of Nguyen and Whitford were re-analyzed and compared with the experimental measurements, [150] 7N2C [151] ), the head is tilted by roughly 6 degrees, [149] relative to the classical orientation of the head. Large-scale tilting has also been found in structures obtained from trans-translocation assays ($ 13 ), [152] as well as in frameshifting-related systems ($ 9 ).…”