Termination of translation in eukaryotes occurs when a translating ribosome encounters a stop codon (UAA, UAG, or UGA) in its A site. This triggers the recruitment of translation termination factors eRF1, a tRNA-mimicking protein responsible for decoding the stop codon and catalyzing peptide release, and eRF3, a translational GTPase that stimulates peptide release in a GTP-hydrolysis-dependent manner. Upon successful stop codon decoding by eRF1, eRF3 carries out GTP hydrolysis and dissociates from the ribosome. eRF1 subsequently gets accommodated into the peptidyl transferase center (PTC) and catalyzes the release of the nascent peptide. The structures for the pre-accommodated eRF1 with eRF3 trapped on ribosome using non-hydrolysable GTP analogs as well as for the PTC-accommodated eRF1 have been solved using cryogenic electron-microscopy (cryo-EM). The structures reveal the binding mode and interactions between the release factors and the pre-termination complex. However, the mechanism of eRF3 GTPase activation and subsequent eRF1 accommodation into the PTC remains poorly understood.
To address this knowledge gap, we used single-particle time-resolved cryo-EM (TRCEM) to capture the structures of intermediates formed during the termination process. For our TRCEM experiments, we first developed a Polydimethylsiloxane (PDMS)-based modular microfluidic mixing-spraying device with a SiO₂ internal coating. The device has a SiO₂-coated PDMS-based 3D splitting-and-recombination (SAR) micro-mixer capable of mixing two fluids within 0.5 ms with more than 90% efficiency. The SiO₂coating strengthens the PDMS channels and acts as a hydrophilic barrier preventing sample adsorption to the PDMS surface. The micro-mixer is connected to a glass microcapillary that acts as the reaction channel. Channels of different lengths can be used to vary the overall reaction time between 10 ms and 1000 ms. The microcapillary is connected to a 3D micro-sprayer for generating a 3D plume of sprayed droplets. A cryo-EM grid is passed through the spray cone to collect droplets and is rapidly plunged into liquid cryogen for vitrification.
By using TRCEM as well as the conventional blotting method for cryo-EM sample preparation, we captured the reaction between a pre-termination (pre-TC) mimic and the ternary complex of eRF1, eRF3, and GTP at reaction times of 450 ms, 900 ms, 15 s, and 10 min. Classification of the cryo-EM data resulted in maps for five distinct factor-bound classes. Four maps belonged to intermediates with densities for eRF1 and eRF3 bound to the pre-TC in varying conformations. The fifth map had a density matching the PTC-accommodated eRF1. Population analysis allowed us to arrange the classes chronologically and track the events leading to GTPase activation during the termination process. Atomic model building and refinement allowed us to determine the hydrolysis state of the eRF3-bound GTP and revealed the catalytic mechanism for GTP-hydrolysis. The models revealed a potential mechanism for GDP dissociation post-GTP-hydrolysis.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/zrrp-s228 |
| Date | January 2024 |
| Creators | Dadhwal, Prikshat |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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