Findings published in Nature revealed how both a smaller and a larger amino acid than alanine are mistranslated.

Scientists from The Scripps Research Institute have uncovered why there are two chemical checkpoints to ensure quality control of alanine in protein production when other amino acids seem to have only one. They figured out the chemical basis for why glycine and serine are often confused for alanine, which led them to conclude that the second check has been put in place to make sure alanine and not glycine or serine is incorporated into a protein.

The study appears in the December 10 issue of Nature in a paper titled “Paradox of mistranslation of serine for alanine caused by AlaRS recognition dilemma.”

When amino acids are put in the wrong order, the resulting mistranslations can have devastating consequences for the health of the organism. Normally, transfer RNAs (tRNAs) transport specific amino acids to the ribosomes so that amino acids can be added to their correct place in a growing chain. For example, the enzyme that adds the amino acid alanine to tRNAs is called alanyl-tRNA synthetase (AlaRS).

Research reported in Nature’s January 2008 and Science’s August 2009 issues by Paul Schimmel, a member of The Skaggs Institute for Chemical Biology at Scripps Research and senior author of the current study, showed that there are two checks for alanine. AlaRS not only loads the tRNA with an amino acid but also checks to make sure it attached the right one. In addition, many organisms, from bacteria to humans, have an extra freestanding protein called AlaXp to ensure that alanine is not confused with other amino acids.

“The editing function is redundant,” notes senior research associate Min Guo, Ph.D., of the Schimmel-Yang lab who is first author of the new paper. “This leads to several questions: Why are the cells so sensitive to alanine mistakes in particular? Why did AlaXps evolve so early? And why are redundant proteins still present that you supposedly don’t need?”

In their latest research, the scientists used a variety of techniques including x-ray crystallography as well as kinetic and mutational analysis to answer these questions. The results showed that one reason for the difficulty AlaRS has in distinguishing alanine from serine and glycine is that the active site on the AlaRS molecule is a large, flexible pocket. Instead of acting as a rigid lock for a single key, the cavity flexes to hold not only its target alanine but also similar-size molecules, serine and glycine.

Serine and glycine are not exactly the same size as alanine, though. That’s where the “serine paradox” comes in. Glycine is smaller, and serine is larger than alanine. Current theory would not predict that molecules that are both larger and smaller than alanine would be a problem for AlaRS.

“The reason we call it a paradox is because none of the other tRNA synthetases have a problem misactivating both a smaller and a bigger amino acid,” says Dr. Guo. “Theoretically, the tRNA synthetases should have the most problem recognizing a smaller amino acid because a smaller one can also go into the binding pocket. The smaller one is easy to understand. Now we explain the bigger one.”

The team found that within AlaRS’ binding pocket the acidic group of Asp235 creates an extra hydrogen bond with the larger serine molecule. This additional bond turns out to be the major force that helps to secure the misplaced serine in the pocket despite its larger size.

X-ray analysis also revealed that Asp235 is critical for holding the amino group of alanine. Attempts by the Schimmel-Yang lab to replace Asp235 with another residue failed. In fact, the scientists found that to make a change that would eliminate the interaction with serine would also impact negatively on the interaction with alanine. They thus suggest that AlaXp exists to provide a second check and eliminate any serine that is attached to tRNA.

Together, AlaRS’ large, flexible pocket and the additional hydrogen bond with serine explain the chemical basis for frequent confusion of glycine and serine for alanine. Hence the need for additional checks to ensure that alanine, not one of its look-alikes, is incorporated into a protein when called for.

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