Finding overturns long held ideas about how cells build proteins

Writing in the May 28 issue of Cell, Johns Hopkins researchers report that four critical components of cells' protein-building machine don't do what scientists had long assumed.

The machine, called the ribosome, is a ball of RNA (DNA's cousin) surrounded by proteins. In the RNA center, genetic instructions are read, the right protein building block is added onto a growing chain, and at the appropriate time the chain is snipped and released.

But while researchers have long known that the ribosome builds proteins, little is understood about exactly how it adds to growing proteins and how it releases the finished product.

In the hunt for these details, scientists have focused on four RNA building blocks, or nucleotides, deep within the machine that are identical in every species, from bacteria to humans. Because they sit where the protein chain is actually built, these "universally conserved" nucleotides in the ribosome were thought to help that process.

Unexpectedly, Johns Hopkins researchers have discovered that these four nucleotides are not important for building the protein, but instead help release the finished product. In laboratory experiments, the researchers found that ribosomes with these key spots changed could put proteins together as well as normal ribosomes, but let go of the finished product much more slowly.

"Most scientists have said that these four nucleotides must be critical for synthesis of the growing protein because of their location, and we fully expected that our studies would prove that to be true," says Rachel Green, Ph.D., associate professor of molecular biology and genetics and a Howard Hughes Medical Institute associate investigator. "We were shocked that they appear to play very little if any role in building proteins, and instead normally speed the protein's release at the right time.

"Our finding underscores the idea that if you build a well-defined system to study a biologic question, you'll get answers you didn't expect," adds Green.

Instead of validating existing ideas about the role played by these conserved nucleotides, the researchers' work suggests a brand new model, says Green. The ribosome actually has another set of evolutionarily unchanged nucleotides, slightly farther from its "business end." Green and her colleagues believe these nucleotides are really responsible for catalyzing the protein's construction, simply by properly orienting the new building block and the chain, an idea they are testing now.

For the current study, graduate student Elaine Youngman first created 12 mutant ribosomes -- the 12 singly changed alternatives to the natural ribosome. (Four nucleotide building blocks are used to make RNA. Each mutant had one of the four conserved nucleotides replaced with one of its three alternatives.)

Then Youngman tested the ability of each of the purified mutant ribosomes to add a molecule called puromycin onto a growing protein chain. Puromycin looks and acts like a normal protein building block, or amino acid, ready for protein synthesis. However, each amino acid normally used by the ribosome has an identifying RNA "tag," which puromycin almost entirely lacks. 

"We had hoped to see one of the mutants really stand out as being incapable of doing this reaction," says Green. "But instead, none of the mutants could do it efficiently, which left us scratching our heads."

So the researchers tested the ribosomes' ability to use their normal starting materials: actual amino acids attached to their correct RNA tag. Much to the researchers' surprise, the mutant ribosomes performed perfectly.

"The key difference between puromycin and the real amino acids used in this reaction is that puromycin lacks the RNA tag," says Green. "Researchers use puromycin all the time to study ribosome function, for many good reasons. But now we know ribosomes don't always treat this molecule as they would real amino acids."

As a result, she says, scientists should carefully evaluate whether the use of puromycin could have skewed interpretation of their experiments.

Amino acids' RNA tags, called transfer RNA or tRNA, help the ribosome identify the right amino acid to add to the protein, since it matches itself to the genetic instructions (messenger RNA) the ribosome is reading. But the tRNA also acts as a handle for the small amino acid: Specific parts of the tRNA are "held" by other evolutionarily unchanged nucleotides in the ribosome as the amino acid is added onto the protein. Green points out that these nucleotides quite likely position the amino acid properly to catalyze what is already a pretty easy reaction.

The scientists were funded by the National Institute of General Medical Sciences and the Howard Hughes Medical Institute. Authors on the paper are Biochemistry and Molecular Biology graduate student Youngman, Green, laboratory technician Julie Brunelle and undergraduate student Anna Kochaniak, all of Johns Hopkins.

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