An artificial ribosome that doesn’t need to fall apart to work

July 29, 2015 § 1 Comment

By joining the RNA of the large and small ribosomal subunits into one molecule, researchers engineered a functional, tethered ribosome (Ribo-T). Ribo-T proves that reversible dissociation and association of ribosomal subunits is not required for efficient protein synthesis.  Credit: Erik Carlson

By joining the RNA of the large and small ribosomal subunits into one molecule, researchers engineered a functional, tethered ribosome (Ribo-T). Ribo-T proves that reversible dissociation and association of ribosomal subunits is not required for efficient protein synthesis.
Credit: Erik Carlson

Frustration has its perks. In a paper just out in Nature, researchers describe making an artificial ribosome because they couldn’t get normal ribosomes to do what they wanted. In creating this artificial ribosome, called Ribo-T, the investigators unwittingly turned conventional molecular biology wisdom on its head: Unlike regular ribosomes, Ribo-T doesn’t need to fall apart and come together again to support protein synthesis.

 

Alexander Mankin from the University of Illinois at Chicago says his group and that of Michael Jewett at the Northwestern University were trying to teach normal ribosomes new tricks, like getting it to translate “difficult-to-make” proteins or to take in unnatural amino acids to make special polymers. “We were frustrated with our inability to test or alter the functions of the ribosome,” says Mankin.

 

Trying to tweak the existing ribosomal RNA, which does much of the work of protein synthesis in the ribosome, didn’t go anywhere. Changes to it killed the cell.

 

So Mankin, Jewett and their teams considered making a portion of the ribosome that would be able to guide the ribosome into making the special polymers. But the problem is that the ribosome, made up of two subunits, falls apart and comes together in every cycle of protein synthesis. How would they stop the re-engineered portion of the ribosome from being swapped out by the normal subunit?

 

That’s when the idea of a tether came in. But “dissociation of ribosomal subunits was believed to be a prerequisite for efficient translation, and it was unclear whether ribosome with the tethered subunits would be functional,” says Mankin. Still, the investigators decided to give it a shot.

 

After many tries, one design worked: the Ribo-T. Mankin, Jewett and colleagues engineered a ribosomal RNA that combined sequences from the two subunits of the ribosome into a single unit. Short RNA linkers separated the two subunit RNAs in the contiguous stretch of nucleic acid.

 

And Ribo-T worked even better than anticipated. Not only did Ribo-T make proteins in a test tube, it also made proteins in bacterial cells that lacked naturally occurring ribosomes and keep the cells alive. Mankin still sounds surprised: “We have created probably the first-ever-on-Earth organism which lived with the ribosome where two subunits are combined into a single entity.”

 

He adds that Ribo-T could pave the way to exploring properties of the ribosome and to make a independent protein-synthesis system in cells that does not interfere with the ribosomes that take care of expression the rest of the cellular proteins. But, for now, the investigators are focusing on what sparked off the whole project in the first place: Getting Ribo-T to carry out the tasks that are difficult for normal ribosomes to do.

 

 

Sniff-n-scratch: How mosquitoes sniff us out

December 5, 2013 § Leave a comment

Image credit: Genevieve Tauxe

Image credit: Genevieve Tauxe

Researchers have figured out how mosquitoes pick up our scents. In a paper just out in the journal Cell, a team led by Anandasarkar Ray at the University of California, Riverside, identified one important class of neurons in mosquitoes that detect our skin odors. The work has implications for developing more effective mosquito traps and repellants in places plagued with mosquito-borne diseases, such as malaria and dengue.

Mosquitoes follow carbon dioxide emitted when we breathe, and, once they get close enough, dive toward our bare skin, attracted by the odors given off there. While the neuron that picked up carbon dioxide had been known, the identity of the neuron or neurons with odor receptors that attracted mosquitoes to human skin scents remained unknown.

Ray says that, for many years, he and his colleagues focused their search for human-skin odor receptors on the complex mosquito antennae, which express numerous members of an olfactory receptor gene family thought to be involved in picking up scents. They ignored the simpler maxillary palp organs, small fingerlike sensory structures, that contain the carbon dioxide receptors. Conventional wisdom was that the carbon dioxide receptor “was narrowly tuned, responding primarily to carbon dioxide,” says Ray.

So imagine the investigators’ surprise when they discovered “the carbon dioxide receptor is an extremely sensitive detector of several skin odorants,” says Ray. “In fact, it is far more sensitive to some of these odor molecules when compared to carbon dioxide.”

The investigators used a variety of techniques, including the use of a wind tunnel filled with mosquitoes flying toward beads coated with a human foot odor, to establish that the carbon dioxide receptor neuron, called cpA, also picked up foot odors.

To see if cpA can be targeted by chemicals that might be developed as mosquito traps, the investigators next screened nearly half a million compounds to find ones that either activated or shut down cpA. They eventually settled on two compounds: ethyl pyruvate, a fruity-smelling cpA inhibitor used as a flavor agent in food, and cyclopentanone, a minty-scented cpA activator used as a flavor and fragrance agent. Ethyl pyruvate substantially reduced the mosquitoes’ attraction toward a human arm, in effect acting as a repellent. Cyclopentanone, meanwhile, lured mosquitoes, so it could be used as an attractant in a trap.

Ray’s group earlier had identified neurons in insects that detect the repellent DEET. That work, explains Ray, can help researchers devise ways to repel mosquitoes. The current work described in the Cell paper can be used to come up with ways to trap mosquitoes or mask the cues that attract them to humans.

Ray already has moved toward developing applications of the latest work. “Parts of this carbon dioxide receptor technology has been licensed to an insect research company called Olfactor Labs hat I helped set up in 2010,” he says. “This research company is performing additional discovery and large-scale screening of the carbon dioxide receptor.” The Kite Patch, a sticker that repels mosquitoes, is one of the company’s products.

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