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.

 

 

Double whammy: Making antibiotics more effective by using inhibitors of nitric oxide synthase

October 21, 2013 § Leave a comment

Nitric oxide inhibitors make antibiotics work more effectively. Image credit to Jeff Holden.

Nitric oxide inhibitors make antibiotics work more effectively. Image credit to Jeff Holden.

It happens repeatedly in science: Research in one area serendipitously is found to have potential applications in another area. In this week’s issue of the Proceedings of the National Academy of Sciences, investigators show that compounds originally developed to treat neurodegenerative disorders could be used to inhibit pathogenic bacteria.

Thomas Poulos’ group at the University of California, Irvine, have been collaborating with Richard Silverman’s group at Northwestern University to develop inhibitors for a protein called nitric oxide synthase, or NOS. These inhibitors were geared to act on NOS found in the mammalian central nervous system.

But in 2009, a paper by another group caught their attention. The authors of this paper had shown that nitric oxide, which is made by NOS, helped pathogenic bacteria resist antibiotics. “This suggested that inhibiting bacterial NOS might improve the killing efficacy of antibiotics,” says Poulos. “Since we had all these unique NOS inhibitors, we decided to test for the ability of these compounds to work synergistically with antibiotics.”

The investigators picked as a model organism Bacillus subtilis, which is much like the pathogenic methicillin-resistant Staphyloccocus aureus (better known as MRSA) and Bacillus anthracis, which causes antrax.

First, the investigators made sure that their NOS inhibitors could work alongside antibiotics to halt the growth of bacteria. Then they took the ones that worked and checked to see if the compounds would bind differently to the bacterial and mammalian versions of NOS. “We obviously want those that favor, or at least bind differently, to the bacterial enzyme,” notes Poulos.

The investigators narrowed their attention to two inhibitors. When bacteria were treated with each inhibitor and an antibiotic, they were killed more effectively than when treated with just an antibiotic. By comparing the crystal structures of the inhibitors bound to bacterial and mammalian NOS, the investigators found that these inhibitors targeted a region in the bacterial NOS that appeared to be particularly susceptible to these compounds.

This finding demonstrates that targeting bacterial NOS is one way to tackle pathogenic bacteria that may be growing more resistant to antibiotics.

But, in the meantime, Poulos says, the NOS inhibitors need to be made more selective. “We now know which region of the enzyme to target, so we ought to be able to develop compounds that selectively bind to bacterial NOS,” he says, adding that the team also needs to test its compounds in animal models.

Postscript: Research into antibiotic-resistant bacteria and new bacterial inhibitors always remind me of this classic song:

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