Direct measurement of the mechanical work during translocation by the ribosome

Abstract

A detailed understanding of tRNA/mRNA translocation requires measurement of the forces generated by the ribosome during this movement. Such measurements have so far remained elusive and, thus, little is known about the relation between force and translocation and how this reflects on its mechanism and regulation. Here, we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules, against an applied force. We find that translocation rates depend exponentially on the force, with a characteristic distance close to the one-codon step, ruling out the existence of sub-steps and showing that the ribosome likely functions as a Brownian ratchet. We show that the ribosome generates ∼13 pN of force, barely sufficient to unwind the most stable structures in mRNAs, thus providing a basis for their regulatory role. Our assay opens the way to characterizing the ribosome's full mechano–chemical cycle.

DOI: http://dx.doi.org/10.7554/eLife.03406.001

eLife digest

Producing a protein first requires its gene to be transcribed into a long molecule called a messenger RNA (mRNA). A complex molecular machine called the ribosome then translates the mRNA code by reading it three letters at a time. Each triplet of letters—known as a codon—tells the ribosome which amino acid to add next into the protein. After adding an amino acid, the ribosome moves along the mRNA molecule to read the next codon and add another amino acid into the protein chain.

While researchers understand how protein chains are formed, how the ribosome shifts along the mRNA strand—a process called translocation—is still unclear. It is known that this process involves many force-generating movements and changes to the shape of the ribosome. However, it is only recently that researchers have been able to measure these forces.

Using optical tweezers—an instrument that uses a highly focused laser beam to hold and manipulate microscopic objects—Liu, Kaplan et al. followed individual ribosomes as they translated an mRNA and measured the effect that applying an opposing force has on the rate of translation. The results shed new light on the mechanism of translocation. First, Liu, Kaplan et al. found that ribosomes jump directly from one triplet to the next in the mRNA sequence, rather than moving there in a series of smaller steps. Next, the results indicate that translocation occurs spontaneously, driven by thermal energy, while chemical reactions prevent the reverse movement, in a mechanism known as a ‘Brownian Ratchet’.

Measurements of the maximum force generated by the ribosome also give insights into how translation is regulated. Strands of mRNA can fold into certain structures that slow down translation, because the mRNA must first be unfolded before the ribosome can translate it. Liu, Kaplan et al. found that the maximum force generated by a ribosome is only just enough to unwind these mRNA structures, making the translation rate highly sensitive to the existence of such structures, and the structures themselves of high importance for regulating transcription.

Given its importance as the ultimate decoder of the genetic information, understanding the ribosome's function and regulation has broad implications. The work of Liu, Kaplan et al. opens the way for a full characterization of the role of mechanical forces in the translation process.

DOI: http://dx.doi.org/10.7554/eLife.03406.002