UMass study reveals importance of second proofreading in amino acid sequence

The reading of mRNA and translation into a functional protein is an intricate process performed by the ribosome with high fidelity.

Research performed by University of Massachusetts Medical School structural biologists Andrei Korostelev, Ph. D., and Anna Loveland, Ph. D., as well as Gabriel Demo, Ph. D., now a group leader at Masaryk University, provided clear evidence that a second proofreading step assists in maintaining high fidelity for the amino acid sequence.

An article in the July 1 edition of Nature revealed how the ribosome incorrectly selected a tRNA but later rejected it, ensuring the incorrect amino acid would not be incorporated into the elongating peptide chain.

The study provided atomic-level images of 17 structures of the ribosome using Cryo-EM microscopy.

Korostelev explained the most significant portion of their work to Current Science Daily.

“I feel we've contributed equally to understanding how the ribosome accurately performs two stages of protein synthesis: elongation and termination,” he said.

“Elongation. The ribosome is a very accurate machine, rarely inserting the wrong amino acid into a protein (a few mistakes per thousands of amino acid insertions), so most proteins have no errors at all. Using new high-resolution cryo-electron microscopy (cryo-EM) technology, we have obtained molecular movies that show how the ribosome cooperates with transfer RNA (tRNAs, which bring amino acids to the ribosome) and elongation factor EF-Tu (the tRNA carrier protein) to ensure that correct tRNAs are selected, and incorrect tRNAs are rejected.

“Termination. When the ribosome reaches the end of a coding region of messenger RNA — mRNA, the genetic instructions of a protein — it stops translating and releases the protein,” Korostelev said. “Using high-resolution X-ray crystallography and cryo-EM methods, we have been able to visualize how the ribosome stops at the end of a coding region and how it uses a helper protein — termed release factor — to release the newly made protein into the cell to perform its function.”

Korostelev said there is a way for people without an extensive background in the field to understand it.

“Decoding is similar to when shopping on Amazon, you quickly select and order an item that looks like the one you want (initial selection of tRNA). GTP hydrolysis is like paying for the item,” Korostelev said.

“Proofreading is like inspecting the item after payment, realizing that something is wrong (e.g., Amazon shipped the wrong color or size), and rejecting the item before using it,” he said. “There are two stages of proofreading: during the ‘return window’ (i.e., while EF-Tu still holds the tRNA), you can send the item back to Amazon; after the return window expires (i.e., after EF-Tu dissociates), you can throw the item in the dumpster.”

He also provided a more technical explanation.

“Textbooks teach us that many processes in biology are binary (e.g., accept or reject) because proteins responsible for these processes can switch between ‘on’ or ‘off’ states,” Korostelev said.

“This switching is often defined by a chemical step called hydrolysis, whereby an ATP or GTP molecule (often called ‘energy molecules,’ but they are actually not) is broken down by a water molecule,” he said. “Hydrolysis helps proteins change shape and thus switch between the ‘on’ and ‘off’ states. A GTP-hydrolysis switch is generally thought to drive accurate decoding: EF-Tu is paired with GTP when it brings a tRNA to the ribosome; and GTP hydrolysis is required for EF-Tu to hand the tRNA over to the ribosome.

“It turns out, however, that the ribosome’s decision to accept or reject the tRNA is not a binary process. Instead, the ribosome continuously checks if the tRNA matches the mRNA codon — the genetic instruction for the next amino acid — before GTP hydrolysis (known as initial selection), after GTP hydrolysis (known as proofreading), and even after EF-Tu departure, as the tRNA moves into position to add the new amino acid to the growing protein (known as accommodation). During initial selection and proofreading, the ribosome uses a common structural principle in its decoding center to check if the tRNA is correct.”

Korostelev said his training led him into studying protein translation.

“With my background in chemistry and interest in detailed biochemical mechanisms, I wanted to contribute to the understanding of a biological process that is central to all life,” he said. “Translation converts genetic information into proteins, the building blocks of life. Being a conceptually simple but chemically complex process, translation was the obvious field for me to dive into.”

There were some completely unexpected developments, Korostelev said.

“Large biological molecules (macromolecules) like ribosomes are very dynamic: they change shape very fast (many times per second), and such rearrangements are essential for function,” he said. “Structural biologists have traditionally tried to capture different conformations by ‘freezing’ macromolecules in crystals and/or by using inhibitors that stall their movements.

“I didn't expect that we would capture many transient conformations (in near-atomic detail) of a process as dynamic as mRNA decoding,” Korostelev said. “So, I was very excited when Anna Loveland, from my lab, showed me the first images of transient conformations that have long eluded observation. We didn’t use inhibitors, yet we resolved many structural changes that reveal how the ribosome reads the tRNA and how the tRNA carrier protein (EF-Tu) moves as the ribosome accepts or rejects the tRNA. Importantly, our cryo-EM studies revealed essential structural changes on the order of a few Angstroms (1 Angstrom is 10-billion times smaller than 1 meter)."

“This technical advance is truly exciting; I liken it to using a new super-telescope to discover a distant galaxy with an Earth-like planet and creatures moving on its surface.”

He said advancing understanding of the process was truly rewarding. It also provided evidence of the ribosome’s true role.

“We were equally excited to see how the ribosome, EF-Tu, and tRNA move during decoding. Previous work relied on computational simulations or on a limited number of structures stalled by inhibitors,” Korostelev said.

“So, it wasn't clear whether simulations reflect what happens in the cell, and some studies made opposing predictions. So visualizing the transient structures by cryo-EM helped to clarify the order of events and reveal novel features: for example, the ribosome holds strongly to a correct tRNA and helps to move the tRNA a large distance from its initial site of binding to the site where its amino acid cargo is added to the elongating protein chain. The ribosome is not a passive ‘spectator.’”