Translation termination in eukaryotes: polypeptide release factor eRF1 is composed of functionally and structurally...

Area of Science:

• Molecular Biology
• Protein Structure and Function
• Genetics

Background:

• Class-1 polypeptide chain release factors (RFs) are crucial for terminating protein synthesis by hydrolyzing peptidyl-tRNA at the ribosomal peptidyl transferase center.
• In eukaryotes, eRF1 not only triggers translation termination but also binds and activates eRF3, a GTPase dependent on both eRF1 and the ribosome.
• The ability to uncouple peptidyl-tRNA hydrolysis and GTP hydrolysis in vitro suggests distinct functional domains within eRF1.

Purpose of the Study:

• To investigate the functional and structural domains of human eRF1 responsible for its dual roles in translation termination and eRF3 activation.
• To determine if eRF1's functions in peptide hydrolysis and GTPase activation are localized to separate domains.
• To explore the implications of domain separation and charge distribution on eRF1's interactions and evolutionary origin.

Main Methods:

• Deletion analysis of the human eRF1 polypeptide chain to identify functionally distinct domains.
• In vivo binding assays to assess interactions between eRF1 domains and eRF3.
• In vitro assays to measure peptidyl-tRNA hydrolysis and eRF3 GTPase activation.
• Calculation of isoelectric points (pI) for different eRF1 domains to analyze charge distribution.

Main Results:

• Human eRF1 comprises two physically separated and functionally distinct domains: a core domain and a C-terminal domain.
• The core domain (N-terminal and middle parts) is responsible for ribosome binding and termination-codon-dependent peptidyl-tRNA hydrolysis.
• The C-terminal domain binds eRF3 in vivo (independently of the core domain) and is required, along with the core domain, for activating eRF3 GTPase activity on the ribosome.
• The core domain has a high pI (9.74) and the C-terminal domain has a low pI (4.23), indicating significant charge asymmetry.
• The positively charged core domain may interact with rRNA and peptidyl-tRNA, suggesting RNA-binding capabilities.

Conclusions:

• Human eRF1 possesses two distinct functional domains: a core domain for peptide release and a C-terminal domain for eRF3 interaction and activation.
• The uneven charge distribution between these domains likely influences eRF1's binding to the ribosome and eRF3, as well as its catalytic activities.
• The structural and functional divergence of these domains supports the hypothesis that eRF1 evolved through gene fusion.