Essential Errors: Advantageous error-prone DNA Translation

Agarose Electrophoresis is an important method of DNA fragment analysis. Image courtesy of Wikimedia Commons.

In the average adult human, half a pound of proteins is turned over daily–destroyed and rebuilt from scratch. Among the many biological processes that occupy an organism throughout its lifetime, protein production ranks among the most intensive. Most forms of life therefore depend on accurate protein production. For instance, in humans, single base pair mutations in DNA blueprints, leading to a change in one amino acid building block, are known to cause diseases ranging from sickle cell anemia to cancer. However, a recent study at Yale has found an exception. Researchers in professor Dieter Soll’s group, working in collaboration with Antonia van den Elzen from professor Carson Thoreen’s group, examined the genomes–the complete set of genetic material in an organism–of thousands of bacterial species and found that parasitic bacteria with reduced genomes lack protein proofreading mechanisms. How is it that these bacteria survive–and even thrive–with chunks of faulty protein production machinery?

Parasites only have to be “good enough”

The parasitic lifestyle may provide an answer. Organisms can be divided into two major lifestyles: free-living or parasitic. Free-living organisms live in open environments–air, soil, water, or ground–while parasitic organisms live inside other organisms. “These lifestyles are fundamentally different because of the difference in competition,” said Sergey Melnikov, a postdoctoral associate who led the project. While a free-living organism competes with others for environmental resources, a parasitic organism does not; the resources available inside its host are plentiful. Without intense competition, parasitic organisms do not face the same evolutionary pressures as free-living organisms. They do not have to be the best in their habitat to survive and reproduce. They merely need to be good enough.

As a result, parasites can tolerate mutations and genomic deletions that weaken their survival ability. As free-living organisms transfer to parasitic lifestyles, they lose small pieces of their DNA over time due to the imperfect nature of DNA replication. Unlike in free-living organisms, these small losses of functionality are not important in the relatively noncompetitive host environment, so the bacteria survive and continue replicating, eventually losing up to ninety-five percent of their DNA. This process, known as “erosive evolution” or “Muller’s Ratchet,” explains why the genomes of parasitic organisms are much smaller than those of the free-living organisms from which they evolved.

Loss of quality control

This erosion of the genome has consequences for the cellular machinery. During this reduction of genetic code, certain features typically thought to be essential for life can be lost, such as the ability of the protein production machinery to proofread the amino acid sequence being generated. After studying 10,423 genomes of 2,277 bacterial species, Melnikov and his colleagues determined that parasitic bacteria with reduced genomes lose mechanisms responsible for protein quality control. “Parasitic life eradicates one important property of cells: accurate processing of genetic information,” Melkinov said.

According to their research, an unusually large percentage of the parasitic bacteria studied had mutations in their protein synthesis quality control system. One such system identified was the aaRSs editing site of tRNA, where messenger RNAs coming from the organism’s DNA are converted to protein. Seventeen percent of the bacteria had multiple mutations in one synthetase, an enzyme that links two amino acids together, and five percent had multiple mutations in several synthetases. Mutations were more common in species with reduced genomes. For instance, only 6.3 percent of species with genome sizes greater than three million base pairs had editing-site mutations, while 83.8 percent of species with genome sizes less than one million base pairs had percent editing-site mutations. These mutations indicate that these species do not correct erroneous amino acids incorporated into the produced protein.

Surviving and thriving with mutations

Although their amino acid sequences may be incorrect, these parasites somehow still survive. To explain this, Melnikov uses an analogy between headphone cords and proteins. “Think of headphones. It is very easy for them to form knots and fold incorrectly, but only one form of the untangled headphones cord is useful to you,” Melnikov said. Just like headphone cords, proteins can misfold into incorrect structures that prevent them from executing their intended function.

To offset the increased possibility of protein misfolding, parasites have elevated amounts of chaperone proteins. These chaperone proteins help guide the folding of the protein into its biologically active form. Consequently, parasitic proteins are still functional despite defects in their sequence. Moreover, protein clumping, a common and catastrophic consequence of misfolding, is less likely.

All of the errors in protein synthesis proofreading actually help the bacteria survive the host’s immune defenses. This is because the errors result in incredible protein diversity. While typical proteins are constructed from twenty stock amino acids, these bacterial proteins were found to have over a hundred additional amino acid variants. The erroneous use of these unnatural amino acids would typically be prevented by the tRNA proofreading system, which is mutated in these bacteria. The result is that every “copy” of a protein produced is slightly different from the next.

The constant flux in protein structure allows the parasite to evade the host immune system. As soon as the host develops immunity–that is, learns to recognize and destroy certain parasite proteins–the parasite changes its identity. These organisms have evolved to perfect their role as parasites.

Leveraging the errors

Researchers are devising novel approaches to fight parasite infection that exploit precisely this property. “What we are doing here is developing a treatment not based on what the parasite has, but based on what the parasite does not have: the ability to proofread amino acids when building proteins,” Melnikov said.

Traditionally, therapeutics target a feature that is present in a specific parasite, but not in the host, in order to selectively destroy the specific parasite. But this knowledge of faulty protein synthesis can now be exploited to create a therapeutic that targets parasites in general. By increasing the availability of unnatural amino acids, the protein synthesis of the parasite could be disrupted without disrupting the protein synthesis of the host, whose proofreading mechanisms would prevent the use of these erroneous amino acids. If the amino acid sequence is sufficiently disrupted so that the proteins can no longer properly fold and perform functions necessary for life, the growth of the parasite could be suppressed without affecting the growth of the host.

The reliance of parasites on chaperone proteins can also be targeted with small molecules that inactivate chaperones. Although this therapeutic approach could impact the chaperone proteins of the host as well, it likely will affect the parasites much more than it will the host, meaning that even a low dose could disrupt the growth of parasites without significantly impacting host growth and survival. Potential targets for these approaches include the parasites responsible for ulcers (Helicobacteri pylori), sexually transmitted diseases (Ureaplasma parvum), and Lyme disease (Borrelia afzelii).

Studying genomes of over 10,000 bacteria has shown that a parasitic lifestyle leads to the degeneration of the genome, severely diminishing quality control systems in protein synthesis. These errors are a parasite’s greatest strength, as its ever-changing composition ensures it avoids the host’s immune response, but they also present a key weakness to be exploited by future therapeutic approaches.