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The Translocation Limbo

06/27/2013
Rachael Moeller Gorman

How do tRNA and mRNA move through the ribosome during translocation? Three new papers catch them in the act. Learn more...


Transfer RNA (tRNA) and messenger RNA (mRNA) must move through a ribosome’s inner channel synchronously or risk a frame shift that could create an aberrant protein. Scientists understand some of the biochemistry behind this process, but at 20 small movements per second, the intermediate connections between the nucleic acids, ribosome, and enzymes have been hard to pin down.

Structures of trapped 70S ribosome–EF-G complexes. Source: Science




But now, 3 papers published today in Science shine a light on this 50 millisecond period (1-3). By capturing crystal structures at several intermediate stages of translocation, scientists have begun to visualize the moving parts of this complex molecular machinery.

“This has been a major goal for over a decade and it’s been very difficult. These dynamic complexes do not want to sit down and wait for you to take their pictures,” said Harry Noller, professor at the University of California, Santa Cruz, whose lab has conducted some of the most fundamental research on ribosomes and ribosomal RNA.

There are four major players that need to be trapped in this translocation dance: ribosomes plucked from the organism Thermus thermophilus, tRNA, mRNA, and the elongation factor EF-G, which catalyzes the movement of the complex through the ribosome. Noller’s lab incubated the first three together, but it was essential to get EF-G, which is a GTPase, to remain attached to the ribosome in order to crystallize it. So Noller and colleagues—as well as the authors of the other studies—incubated EF-G with either the non-hydrolyzable GTP analogue GDPNP or the antibiotic fusidic acid, both of which prevent EF-G release from the ribosome.

Once the team caught the ribosome in the act of translocating, as determined via biochemical assay, the next step was to crystallize the complex with vapor diffusion, which took 10 days. Once harvested, the crystals were flash frozen in liquid nitrogen and then scanned with X-rays. The diffraction data was indexed, integrated, and scaled.

Previously, Noller’s group had shown that the amino acid end of the tRNA first moves along the large subunit, while the other end stays put in a so-called hybrid state. This step involves rotation of the ribosome’s 30S subunit body and can happen spontaneously. But in the next step, shown in the current paper, the movement of mRNA and tRNA through the ribosome, requires energy; The 30S subunit head rotates 15-18 degrees. “The head rotates the tRNA and the mRNA moves with it. And that is how the second step happens,” said Noller.

If the mRNA and tRNA aren’t perfectly synchronized, codons will be translated incorrectly, resulting in a potentially non-functional or toxic protein. Noller found that two universally conserved ribosomal RNA (rRNA) bases that are intercalated between bases of mRNA may act like pawls in a ratchet's gear to stop translocation from going backwards.

In the other papers, David Tourigny, Nobel-prize winner Venkatraman Ramakrishnan, and colleagues at the MRC Laboratory of Molecular Biology in Cambridge report a crystal structure of a ribosome in another intermediary state during translocation, and Arto Pulk at UC, Berkeley and colleagues describe additional intermediate crystal structures in different versions of ribosomal subunit rotation, thus filling in the picture.

“What we have now are a number of structures at key points of the translocation cycle,” said Noller. “You could say we have four different states now. While Disney would want about 400 structures, you can now begin to see the movie of translocation come to life.”

References

1. Zhou, J., L. Lancaster, J.P. Donohue, and H.F. Noller. 2013. Crystal structures of EF-G–ribosome complexes trapped in intermediate states of translocation. Science 340. Published online 06/27/2013.

2. Tourigny, D.S., I.S. Fernández, A.C. Kelley, and V. Ramakrishnan. 2013. Elongation factor G bound to the ribosome in an intermediate state of translocation. Science 340. Published online 06/27/2013.

3. Pulk, A., and J.H.D. Cate. 2013. Control of ribosomal subunit Rotation by Elongation Factor G. Science 340. Published online 06/27/2013.