Old Molecules Yield New Secrets

By Jason Socrates Bardi

Ancient structures can have many lives.

The Tower of London, for instance, was in the 15th, 16th and 17th centuries an infamous prison where the likes of Thomas More, Anne Boleyn and the sons of Edward IV were executed. In the 18th century, it became an innocuous government building, housing the Royal Mint and pressed silver coins. Today, it is a tourist destination where travelers can get a glimpse of the crown jewels and view displays of the building's 1000-year-old history.

Displays of history exist in biology as well, such as in the architectures of transfer RNA (tRNA) and aminoacyl-tRNA synthetase proteins. These ancient tRNA and aminoacyl-tRNA synthetase molecules are ubiquitous in nature. As we know them today, they are involved in one of the most fundamental processes in life—the culminating step in the expression of a gene whereby nucleotide bases are translated into amino acid proteins.

In the late 1980s, Professor Paul Schimmel, who is the Ernest and Jean Hahn Professor and Chair of Molecular Biology and Chemistry and is a member of The Skaggs Institute for Chemical Biology at Scripps Research, suggested that these molecules may have been involved in a "second genetic code" (a term coined by Nobel prize winner Christian de Duve in a Nature commentary in 1988). This second genetic code would have acted as an earlier method of information storage that is perhaps more appropriately termed the operational RNA code, says Schimmel.

The operational RNA code would have related sequences of RNA to amino acids, allowing RNA enzymes to grab amino acids and "borrow" their chemical structures without guidance from a gene. Amino acids have the ability to catalyze a larger range of reactions than nucleotides, and perhaps early RNA life forms evolved ways to use amino acids and their catalytic abilities. It's also plausible that ancient tRNA-like molecules would be loaded with amino acids by the action of another RNA enzyme and these 'loaded' RNAs would condense together to make peptides.

Now, two back-to-back papers in the latest issue of Molecular Cell from Schimmel, Senior Staff Scientist Manal A. Swairjo, Kellogg School of Science and Technology alumna Martha Lovato, and several of their colleagues at Scripps Research explore the details of how an ancient, operational genetic code could have worked.

High-Resolution Structure and Interdomain Flexibility

The papers provide new insight into how the recognition of tRNA is achieved.

In the process of attachment of amino acids to tRNA molecules, one of 20 different synthetases binds to its corresponding amino acid, a molecule of ATP, and one of 20 different tRNA molecules. The synthetase then hydrolyzes the ATP, attaches the amino acid to the end of the tRNA, and lets go. The basic mechanics of this process have been known for a number of years, but have not been understood in detail. This is particularly vexing for the alanyl-tRNA synthetase, the synthetase that attaches the amino acid alanine to tRNA molecules cognate for alanine, as it is one of the most well-studied proteins in biology.

Until now, there has not been a high-resolution structure of the alanyl-tRNA synthetase molecule that could give insight into how the recognition of tRNA is achieved. In the first paper, applying the technique of x-ray crystallography to the protein from the ancient marine bacterium Aquifex aeolicus, Schimmel and Swairjo solved the structure to high resolution—2.14 Angstroms.

This structure of the alanyl-tRNA synthetase protein docked to tRNA shows how this 453 amino acid protein reads the identity of the tRNA molecule so that it can specifically attach alanine to it. The synthetase reads a single base pair located on one arm of the L-shaped tRNA called the "acceptor stem."

The acceptor stem is the more ancient part of the tRNA molecule—a remnant of the ancient molecules of the second genetic code. In fact, in many cases the other arm of the tRNA L (which carries the information for the amino acid) can be completely removed and the tRNA will still accept its cognate amino acid.

The second paper describes how the system adapted throughout evolution to subtle changes in the RNA. In most organisms the acceptor-stem base pair that is recognized by alanyl-tRNA synthetase is what is referred to in biology as a "wobble" base. It has an unusual hydrogen bonding pattern and forms between a G and a U nucleotide (rather than the standard G-C or A-U pairs).

This wobble base pair is located three base pairs from the acceptor end of the tRNA molecule. But occasionally, as in the mitochondria of the fruit fly Drosophila melanogaster, the recognition base pair is in another spot, only two base pairs from the acceptor end.

The unusual thing about a Drosophila mitochondrion is that its alanyl-tRNA synthetase molecules are not so different from the canonical ones of Aquifex aeolicus and other bacteria. One might expect that they would be, since these molecules are very sensitive to the position of the base pair.

To account for this difference, Drosophila evolved to use a different mechanism to recognize the pair in its new location. Lovato, Schimmel, and their colleagues found that Drosophila alanyl-tRNA synthetase has a 27 amino acid insertion that allows it to shift its recognition to the second base pair. The crystal structure demonstrates how a hinge at the site of the insertion allows the part of the synthetase molecule, which reads the wobble base pair to move freely up and down the acceptor stem of the tRNA molecule.

This "interdomain flexibility" is the first example of adaptive recognition of tRNA identity by synthetases in evolution. Predating the Tower of London by billions of years, alanyl-tRNA synthetase yet insisted on preserving the identity of its cognate tRNA even more rigorously.

The article, "Alanyl-tRNA Synthetase Crystal Structure and Design for Acceptor-Stem Recognition" was authored by Manal A. Swairjo, Francella J. Otero, Xiang-Lei Yang, Martha A. Lovato, Robert J. Skene, Duncan E. McRee, Lluis Ribas de Pouplana, and Paul Schimmel and appears in the March 26, 2004 issue of Molecular Cell. See: http://www.molecule.org/content/article/abstract?uid=PIIS1097276504001261

The article, "Positional Recognition of a tRNA Determinant Dependent on a Peptide Insertion" is authored by Martha A. Lovato, Manal A. Swairjo, and Paul Schimmel and appears in the March 26, 2004 issue of Molecular Cell. See: http://www.molecule.org/content/article/abstract?uid=PIIS109727650400125X


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Structural model of adaptive recognition of tRNA identity by alanyl-tRNA synthetase.