Within cellular micro-machines called ribosomes, chains of genetic material called messenger RNAs (mRNAs) match up with corresponding transport RNAs (tRNAs) to create sequences of amino acids that exit the ribosome as proteins. The incomplete proteins are called nascent chains and are left attached to the ribosome.

Scientists know that some of these nascent strands can regulate ribosome activity and that nascent strands can sometimes interfere with antibiotics—many of which work by targeting bacterial ribosome activity. Scientists do not know why this happens, mainly because it is difficult to visualize what ribosome-peptide-drug interactions look like while incomplete proteins are still attached to the ribosome.

Now, scientists at the University of Illinois at Chicago are the first to report a method for the stable binding of peptides to RNA, which has allowed them to gain fundamental new insights into ribosome function by defining the structures at the atomic level of ribosomes and the shapes that these. Take peptides inside the ribosome.

Their method was recently reported in the magazine nature chemistry.

said Yuri Polikanov, assistant professor in the Department of Biological Sciences at the College of Arts and Sciences. “Until the advent of this new method, we were essentially blinded to seeing what was happening at the active site of the ribosome at this critical moment in time.”

Polikanov and his colleague Egor Syroegin, a doctoral candidate in biological sciences at UIC, used a method called chemical native splicing to combine custom peptides with tRNA to produce what’s called peptidyl-tRNA.

“Obtaining tRNA molecules bound to peptides, similar to those inside the ribosome during protein synthesis, has been a dream of many researchers in this field for nearly two decades,” Polikanov said. “This has been very difficult because there are no enzymes that can bind peptides directly to tRNA.”

“This method has been used for a long time in chemistry, but has never been applied in this way. It mimics nature, basically, and with our advanced imaging expertise we now see how nature works with high precision,” Syroegin said.

With this novel approach, Polikanov and Syroegin identified a set of high-resolution structures of ribosomes that carry peptidyl-tRNAs of different lengths.

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

“We have seen that depending on the sequence, different peptides can form different shapes or folds within the ribosomal tunnel, and we can synthesize different peptides from different sequences and then trace their shape very precisely, due to the high resolution of our structures,” Syroegin said. And now, we can say with great confidence that ‘these peptides, of this sequence, have this shape’ or ‘another peptide having another shape. “This is important because the folding of the emergent peptide determines whether or not drugs will stop the ribosome.”

“This method opens up countless avenues for structural and functional studies aimed at understanding the mechanisms of ribosomal action, as well as sequence-specific ribosomal arrest caused by certain antibiotics,” said Polikanov.

Polikanov and Syroegin are co-authors of the paper, “Insight into Ribosome Function from Structures of Unarrested Nascent Ribosome Chain Complexes,” along with Elena Alexandrova, Research Specialist in the Department of Biological Sciences at UIC.

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

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