Tag: tRNA

Studying tRNAs by cryo-EM, biophysics, and computational modeling

Studying tRNAs by cryo-EM, biophysics, and computational modeling

In a pioneering study entitled “Determining the effects of pseudouridine incorporation on human tRNAs” published in EMBO Journal (Link 1), researchers from Malopolska Centre of Biotechnology (Link2) of the Jagiellonian University in Krakow (Link 3), in collaboration with scientists from the International Institute of Molecular and Cell Biology (IIMCB) in Warsaw (Link 4) and institutions from the United Kingdom and France, have significantly advanced our understanding of how specific modifications in transfer RNAs (tRNAs) affect their structure and stability.

tRNAs are essential molecules decoding genetic information into proteins, fundamental to all living organisms. We know tRNAs as long as we know DNA, but historically structural studies of tRNAs have been challenging due to their small size and complexity. The published work shows the structure of four human tRNAs before and after the enzyme-mediated introduction of pseudouridine (Ψ), linking these modifications to enhanced stability and certain structural changes. The findings reveal that the incorporation of Ψ, which is also incorporated in recent mRNA vaccines, not only stabilizes tRNAs, but also induces significant local structural changes. In detail, interactions between the D- and T-arms of the tRNA were identified as critical for maintaining their overall tertiary structure.

“This study demonstrates the profound impact of RNA modifications on tRNA stability and function,” said prof. Sebastian Glatt (Link 5), the leader of this study. “Our findings not only enhance our understanding of tRNA biogenesis but also highlight the potential applications of engineered tRNAs for therapeutic purposes.” adds Anna Biela, the first author of the work.

Key results from the study include:

• The first cryo-EM structures of multiple unmodified and pseudouridylated human tRNAs.

• Clear evidence that specific pseudouridine modifications significantly increase the stability of tRNAs.

• Context-dependent impact of modifications, indicating that not all pseudouridine modifications are equally beneficial.

The study was possible owing to the interdisciplinary combination of experimental and computational analyses. It utilized the advanced single-particle cryo-electron microscopy (cryo-EM) infrastructure at the Solaris Synchrotron in Krakow (Link 6), the Structural Biology Core Fcaility (SBCF, Link 7) at MCB, novel biophysical techniques developed by Jakub Novak, and computational modeling and simulations.

Read more on SOLARIS website

Grabbing a tRNA by the Tail

Grabbing a tRNA by the Tail

Transfer RNAs (tRNAs) are RNA molecules used by all forms of life, from bacteria to plants to humans, to transfer amino acids to growing protein molecules that have been coded by DNA and transcribed into messenger RNA (mRNA) for translation by ribosomes into proteins. This is one of the most basic, crucial processes of life.

However, tRNAs do more than just perform this essential function and are known to have regulatory roles in translation, transcription, stress response, and even immunity, via specific interactions with a wide array of cellular molecules. Disruption of these interactions has also been shown to be associated with some types of neurological disease and cancer, making it critical to understand how proteins in the cell recognize tRNAs.

Many different proteins have been shown to interact with tRNAs via known protein structural motifs. One of these is the oligonucleotide/oligosaccharide-binding (OB)-fold that has a highly conserved β-barrel structure found in organisms across all domains of life. However, the details of its interactions with tRNA are not completely understood. Recent research from a team at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) has provided new, previously unrecognized, insights into how the OB-fold recognizes the 3’ tail of tRNA molecules and how these interactions impact the function of tRNAs.

The research team used X-ray diffraction data collected at the South East Regional Collaborative Access Team (SER-CAT) beamline 22-ID of the Advanced Photon Source, a U.S. Department of Energy (DOE) user facility at DOE’s Argonne National Laboratory.

The protein structure work in this project focused on a superfamily of proteins called the tRNA binding domain (TRBD) family. These proteins interact with tRNAs via an OB-fold domain that consists of five β strands that form a barrel structure with an α-helical cap. TRBD proteins are found in many different organisms and, while they don’t always have high levels of amino acid sequence conservation, they all contain the OB-fold.

This work started with a TRBD protein from the bacteria Aquifex aeolicus called Trbp111 that is known to bind to many tRNAs. Solution of a new 2.3 Å crystal structure of Trbp111 showed that the protein forms an unusually stable homodimer with the two β-barrels stabilized by a very strong dimer interface. This is consistent with what is known about Trbp111, as A. aeolicus thrives at high temperatures (~90°C) and is also resistant to many common laboratory protein denaturing procedures, suggesting that this type of stable interface may provide a model for artificial protein design and structure-based drug design efforts.

Read more on Argonne website

Image: The figure shows how the OB beta barrel uses its two protruding loops as “pincers” to capture the terminal CA dinucleotide of the tRNA in various representations (tRNAs are shown in green in each panel).