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Optical tweezers pick up the ribosome beat

Thursday, April 3, 2008
Written by Branwyn Wagman

Schematic diagram of ribosome moving along a strand of mRNA held by optical tweezers. Source: Laura Lancaster

For the first time, researchers have glimpsed the physical steps of the ribosome machine as it translates messenger RNA into a protein. Their findings? Protein synthesis has a beat: bop bop bop pause; bop bop bop pause... The three bops correspond to the ribosome reading one codon of mRNA—the three nucleotide bases that instruct the ribosome to add one amino acid to a protein chain. Since the mRNA is so small, on the nanoscale, it takes a tiny apparatus called optical tweezers to measure the pulse.

Harry Noller, the Sinsheimer Professor of Molecular Biology at UCSC, has been studying the ribosome for more than 30 years. His latest findings are the result of a six-year collaboration between three groups within the California Institute for Quantitative Biosciences (QB3): Noller and postdoc Laura Lancaster along with two UC Berkeley labs, those of biophysicist Carlos Bustamante and biophysical chemist Ignacio Tinoco, Jr.

“This is a collaboration between the tweezers guys at Berkeley and the ribosome guy at UCSC,” Noller said. “We got together to figure out if we could use this approach to measure forces exerted by ribosomes during protein synthesis as it moves along the mRNA.” Their research is featured on the cover of the April 3 edition of Nature.

“Tweezers are used to grab single molecules and measure events that are taking place. Measuring forces. Measuring distance. Measuring speeds of things like DNA replication, RNA transcription, and movement of microtubules,” Noller said.

In this research, the three labs asked, “What are the forces the ribosome exerts when it translates mRNA?” Noller added, “We can then break those forces down. If multiple events are happening, we can potentially de-convolute them and start to figure out what they are. It is like putting a stethoscope to the RNA.”

The Noller lab engineered ribosomes containing messenger RNA molecules with strands of DNA attached to both ends to serve as "handles." The DNA strands, in turn, are attached to tiny beads. Both beads are fixed by lasers in the optical tweezers apparatus, and the lasers at each end exert opposing forces on the translation system. To discern the beat, they graphed changes in the force and the distance between the beads as the ribosome traveled along the mRNA to decode it.

Noller said, “mRNA is made of nucleotides, A, U, C, and G, that go through the ribosome one codon at a time. The distance of one or even three nucleotides was at the edge of our ability to measure with tweezers. Our strategy was to put a double helix into the message. In order for the message to move through, it’s got to unzip the helix. So when the ribosome moves along the mRNA by a codon—three nucleotides—it unzips three base pairs, and the apparatus moves by a distance of six nucleotides, not three. This is enough to crack through the threshold of the machine’s sensitivity.”

As the ribosome translates the mRNA to protein, the tweezers apparatus measures the length of the mRNA-DNA construct. When the extension is graphed against time, discrete steps appear. “The ribosome moves along the message in a series of stop-and-go events. Stalling and translocation. Pause–translocate–pause,” Noller said. Each movement corresponds to unzipping the hairpin.

Graphing the distance between the steps yields peaks at 2.7-nm intervals, which corresponds to the length of six nucleotides along an mRNA. So the breaking of three hairpin base pairs accompanies each translocation step. “You can fit the translocation time data to different distributions, and when you do, it fits best with a three-event distribution—three events per jump.”

Noller's research group was the first to solve the complete structure of a ribosome using x-ray crystallography, which was the first step in answering the bigger question: how does it operate?

“Up until now, we were limited to watching a few trillion ribosomes, and they are not synchronized. The details are blurred. Now we can watch one ribosome,” he said.

Noller, who directs the Center for Molecular Biology of RNA at UCSC, said the next phase in this project involves refining the analysis. “We have not yet directly measured the forces exerted by the ribosome. In the experiments in this paper, the forces are ones that the tweezers exert on the ends of the mRNA, and what we are measuring is usually extension of the ends of the mRNA with time, with constant or varying force.”

“Now we have a more sensitive tweezers set up at UCB, and we have devised a way of grabbing a ribosome with one tweezers and the message with the other.” This gives the more direct measurement Noller desires, “We can feel the ribosome pulling on the message.”

The first author of the Nature paper is Jin-Der Wen of UC Berkeley. In addition to Noller, Lancaster, Bustamante, and Tinoco, the coauthors include Courtney Hodges of UC Berkeley; Ana-Carolina Zeri of the Brazilian Synchrotron Light Laboratory; and Shige Yoshimura of Kyoto University.

The California Institute for Quantitative Biosciences (QB3) is a cooperative effort among three campuses of the University of California—Santa Cruz, Berkeley, and San Francisco—and private industry.