UIC scientists use native chemical ligation to fuse peptides with tRNAs

Inside tiny cellular machines called ribosomes, strings of genetic material called messenger RNAs (mRNAs) combine with corresponding transfer RNAs (tRNAs) to create sequences of amino acids that exit the ribosome as proteins. Unfinished proteins are called nascent chains and they remain attached to the ribosome.

Scientists know that some of these nascent chains can regulate ribosome activity and that nascent chains can sometimes interfere with antibiotics, many of which work by targeting the activity of bacterial ribosomes. Scientists don’t know why this happens, mainly because it’s hard to visualize what ribosome-peptide-drug interactions look like while unfinished proteins are still attached to the ribosome.

Now, scientists at the University of Illinois at Chicago are the first to report a method of stable attachment of peptides to tRNAs, which has given them fundamental new insights into ribosome function by determining the atomic-level structures of ribosomes and the forms these peptides take inside the ribosome.

Their method has just been reported in the journal natural chemistry.

“The challenge was to see the structure of the ribosome and the exit tunnel up close in the presence of the nascent peptides because, in nature, the ribosome is very fast for us to capture images or conduct experiments,” said Yury Polikanov. , Associate Professor in the Department of Biological Sciences at the College of Liberal Arts and Sciences. “Until the advent of this new method, we have essentially been blinded to seeing what is happening in the active site of the ribosome at this critical point in time.”

Polikanov and his colleague Egor Syroegin, a UIC PhD candidate in biological sciences, used a method called native chemical ligation to fuse custom peptides with tRNA to produce what is called a peptidyl-tRNA.

“Achieving peptide-bound tRNA molecules, similar to those inside the ribosome during protein synthesis, has remained the dream of many researchers in the field for nearly two decades,” Polikanov said. . “It was extremely difficult because there are no enzymes that can directly attach peptides to a tRNA.”

“The method has been used for a long time in chemistry, but it has never been applied in this way. It basically mimics nature, and thanks to our advanced imaging experience, we now see how nature works at high resolution. “Syroegin said. said.

With this new approach, Polikanov and Syroegin determined a set of high-resolution structures of the ribosome carrying peptidyl-tRNAs of different lengths.

Detailed analysis of these structures provides new and surprising insights into the mechanism of the ribosome catalytic center and answers several long-standing fundamental questions in the ribosome field, Polikanov said.

“We have seen that depending on the sequence, different peptides can form different shapes or folds in the ribosomal tunnel, and we can synthesize different peptides of different sequences and then follow their shape very precisely, due to the high resolution of our structures,” Syroegin said. “So now we can confidently say ‘these peptides, of this sequence, have this shape’ or ‘another peptide has another shape.’ This is important because the folding of nascent peptides determines whether drugs would stop or not the ribosome.”

“This method opens countless avenues for structural and functional studies aimed at understanding the mechanisms of ribosome function, as well as the sequence-specific ribosome blockade induced by certain antibiotics,” Polikanov said.

Polikanov and Syroegin are co-authors of the article, “Insights into the ribosome function from the structures of non-arrested ribosome nascent chain complexes”, with Elena Aleksandrova, research specialist in the Department of Biological Sciences at UIC.

This work was supported by the National Institutes of Health (R01-GM132302, R21-AI163466), National Science Foundation (MCB-1907273), and Illinois State Startup Funds. This work is based on research conducted on the Northeast Collaborative Access Team beamlines at the Advanced Photon Source at Argonne National Laboratory (DE-AC02-06CH11357).