4. Aug. 2020
Researchers from the University of Massachusetts Medical School, including Slovak structural biologist Gabriel Demo (now Research Group Leader at CEITEC MU), developed an approach to visualise protein synthesis with time resolved Cryo-electron microscopy (cryo-EM), and revealed the translation accuracy in unprecedented detail. Previously, it was only possible to see snapshots of different parts of the process that was stalled by inhibitors. Their method could become the new gateway for the visualisation of complex biochemical pathways without inhibitors. The researchers’ study was recently published in the prestigious scientific journal, Nature.
Ribosomes are the molecular machines found within all living cells, and play a key role in biological protein synthesis (mRNA translation). They read genetic instructions and translate the instructions into proteins. This process is responsible for the translation of certain parts of DNA into a particular trait. In the cell, ribosomes can connect more than ten amino acids per second. Therefore, seeing a complete reaction of amino-acid-bound tRNA selection at near-atomic detail has represented a big challenge.
The research team cooled all of the components of the reaction on ice before the experiment and used real time-resolved cryo-EM. This technique allowed them to observe what happens during an entire reaction. The team was the first to visualise the stages in which ribosomes synthesise life-sustaining proteins without using any inhibitors, which is comparable to the actual process happening in a cell. This technique can be also applied to other challenging reactions that have been previously studied, with the help of inhibitors.
The researchers used the ground-breaking technology called “cryo-EM” for visualising the delivery of amino-acid-bound tRNA to the ribosome at different time points. Visualising the structural ensembles provided an unprecedented view of the complete reaction, from the initial selection, to transfer RNA (tRNA) proofreading, and the addition of an amino acid to the growing protein. The main difference between this study and previous work on this topic is that the researchers were able to observe more detailed states that weren’t previously visible. “These structural ensembles, including short-lived transient states, are critical because they are the ones in which decision-making takes place for the ribosome to accept the correct tRNAs, and to reject the incorrect ones,” explained Korostelev, the corresponding author of the study. The results indicate that time-resolved cryo-EM could become the ultimate structural biochemistry method for visualising complex biochemical pathways without inhibitors.
The research was performed at the University of Massachusetts Medical School by structural biologists, Anna Loveland and Gabriel Demo, and led by Research Group Leader, Andrei Korostelev. Gabriel Demo, one of the authors of this study (formerly a postdoctoral researcher in the Korostelev Lab) is now a Research Group Leader at CEITEC Masaryk University. His research group is investigating the mechanistic details and functional outcomes of coupled transcription-translation. He uses an integrative approach that combines proteomics, molecular biology, biochemistry, and structural biology. Demo’s research could significantly improve the understanding of gene regulation in bacterial and virus-infected mammalian cells. Demo’s research group is CEITEC’s newest acquisition, perfectly complementing the expertise of other excellent structural biologists at CEITEC, such as ERC grant holder Pavel Plevka and Richard Stefl, and ERC-CZ grant holder Robert Vacha.
Watch the protein synthesis visualisation (to play the video, click on the picture below):
Author: Ester Jarour