Study and visualize RNA translation mechanisms at the nanoscale

Figure (top) shows the activity of a ribosome across an RNA molecule. As the ribosome translates the RNA fragment, it becomes shorter and the end-to-end distance between the two beads lessens proportionally. This enables us to measure the activity and states of translation proteins as well as the kinetics and formation of nascent proteins.

Also, multicolor fluorescence detection allows quantifying the FRET efficiency changes of a labeled ribosome in time. In this way, we can correlate a certain conformational change of the ribosome with its activity bursts (Figure, bottom).

Figure 1 shows the results of an experiment where we stretched and relaxed the RNA molecules with a constant load (10 nm/s, blue) while recording the force-extension curves. At approximately 8 pN we observe an unfolding rip, approximately 15 nm long. When we relaxed the molecule the refolding curve followed the same path as the unfolding, which corresponded to a rip of similar size. This shows that the unfolding of the RNA molecule is a reversible process. Cycles of unfolding and refolding could be repeated multiple times with the same result.

In the second experiment, we evaluated the structural transitions of our RNA target molecule over time. To this end, we brought a single RNA molecule to a specific pretension and studied its transitions. Figure 2 shows two major states at tensions around 6.2 pN and 7.2 pN, while a third conformational state was also occasionally accessed.

‍

Sample courtesy of Prof. Hang Shi at Tsinghua University.