It’s a fundamental rule governing how the genetic code works. Ribosomes, the factories that assemble proteins in all types of living cells, read three letters (or nucleotides) of messenger RNA at a time.

In some instances, the ribosome can bend its rules, and read either two or four nucleotides, altering how downstream information is read. Biologists call this normally rare event ribosomal frameshifting. For an ordinary gene, the event of a frameshift turns the rest of the ensuing protein into nonsense. However, many viruses exploit frameshifting, because they can then have overlapping genes and fit more information into a limited space.

Regulated frameshifting takes place in human genes too, and understanding frameshifting is key to recent efforts to expand the genetic code. Researchers are aiming to use the process to customize proteins for industrial and pharmaceutical applications, by inserting amino acid building blocks not found in nature.

“Going back to the 1960s, when the genetic code was first revealed, there were many studies on ribosomal frameshifting, yet no-one really knows how it works on a molecular and mechanistic level,” says Christine Dunham, PhD, assistant professor of biochemistry at Emory University School of Medicine. “What we do know is that the ‘yardstick’ model that appears in a lot of textbooks, saying that the anticodon loop dictates the number of nucleotides decoded, while elegant, is probably incorrect.”

Dunham, who first studied the topic as a postdoc, and her colleagues published a paper this week in PNAS where they outline a model for how ribosomal frameshifting occurs, based on structural studies of the ribosome interacting with some of its helper machinery. Co-first authors of the paper are postdoctoral fellows Tatsuya Maehigashi, PhD and Jack Dunkle, PhD.

If mutations add or subtract a letter from a protein-coding gene, thus disabling it, then researchers have found there are compensatory mutations that can push the genetic letters back into the correct frame.

Some of these are simple enough: they’re just second mutations in the protein-coding gene itself that shift the reading frame back. However, others are separate, within the machinery for translating the genetic code, namely tRNAs: the adaptors that bring amino acids into the ribosome for protein synthesis. So-called “suppressor” tRNAs can compensate for a forward frameshift in another gene. Some suppressor tRNAs have an extra nucleotide in the anticodon loop, the portion that pairs up with the messenger RNA. This observation led to the yardstick model.

“What led the field to discard the yardstick model was: finding other suppressor tRNAs that compensate for the frameshift in different ways, besides sticking an additional nucleotide into the anticodon loop,” Dunham says.

Dunham, Maehigashi and Dunkle chose to examine a suppressor tRNA that has an extra nucleotide in the anticodon loop, but on the opposite side from where it would need to be in the yardstick model (see diagram).

Normal tRNA on left, suppressor tRNA on right. The red G is not in the position suggested by the yardstick model.

Yet it still works to shift the ribosomal reading frame back efficiently. Through X-ray crystallography, the researchers were able to map how this suppressor tRNA interacts with the ribosome and the messenger RNA on a molecular level.

“Unexpectedly, we found the insertion alone does not cause the frameshift, rather it is the larger anticodon loop that distorts a highly conserved feature of the tRNA while still retaining its ability to recognize the mRNA codon,” Dunham says. “We think this disruption causes the tRNA to not be recognized properly by other translation factors, thus causing a shift in the frame.”

“It is known that tRNAs are flexible. They have to interact with a variety of enzymes during the translation cycle, and undergo large conformational changes as they traverse their binding sites in the ribosome. What we have found is that this mutant tRNA has a novel structure while bound to the ribosome; it adopts this conformation at all costs to preserve its normal interaction with the mRNA.”

Many researchers have become interested in “expanding the genetic code,” or incorporating amino acids into proteins that are not normally found in nature, with a host of applications in biotechnology. With limited success, they have been using suppressor tRNAs as the vehicles for the unnatural amino acids. The Dunham lab’s findings may provide insights to increase the efficiency of this process, she says.