Translation termination in eukaryotes: Polypeptide release factor eRF1 is composed of functionally and structurally distinct domains

Abstract

Class-1 polypeptide chain release factors (RFs) trigger hydrolysis of peptidyl-tRNA at the ribosomal peptidyl transferase center mediated by one of the three termination codons. In eukaryotes, apart from catalyzing the translation termination reaction, eRF1 binds to and activates another factor, eRF3, which is a ribosome-dependent and eRF1-dependent GTPase. Because peptidyl-tRNA hydrolysis and GTP hydrolysis could be uncoupled in vitro, we suggest that the two main functions of eRF1 are associated with different domains of the eRF1 protein. We show here by deletion analysis that human eRF1 is composed of two physically separated and functionally distinct domains. The "core" domain is fully competent in ribosome binding and termination-codon-dependent peptidyl-tRNA hydrolysis, and encompasses the N-terminal and middle parts of the polypeptide chain. The C-terminal one-third of eRF1 binds to eRF3 in vivo in the absence of the core domain, but both domains are required to activate eRF3 GTPase in the ribosome. The calculated isoelectric points of the core and C domains are 9.74 and 4.23, respectively. This highly uneven charge distribution between the two domains implies that electrostatic interdomain interaction may affect the eRF1 binding to the ribosome and eRF3, its activity in the termination reaction and activation of eRF3 GTPase. The positively charged core of eRF1 may interact with negatively charged rRNA and peptidyl-tRNA phosphate backbones at the ribosomal eRF1 binding site and exhibit RNA-binding ability. The structural and functional dissimilarity of the core and eRF3-binding domains implies that evolutionarily eRF1 originated as a product of gene fusion.

Omnipotent decoding potential resides in eukaryotic translation termination factor eRF1 of variant-code organisms and is modulated by the interactions of amino acid sequences within domain 1

“…The cDNA encoding the full-length human eRF1 was inserted into the NdeI-XhoI sites of the expression vector pET23b(ϩ) (Novagen). The human eRF1 containing a His-tag at the C terminus was expressed in Escherichia coli and purified by using a metal affinity column, as described (7,11). An Sp-eRF1 overexpression plasmid was constructed by subcloning the NcoI-NheI segment of Sp-eRF1 into the NcoI-BamHI sites of pET15b (Novagen) by linker ligation to give rise to pET15b-Sp-eRF1.…”

“…This domain assignment relies on the assumptions that the universal GGQ motif (7) located at the tip of domain 2 is a structural counterpart of the tRNA aminoacyl group on the CCA-3Ј acceptor stem, and that domain 1, in which a codonspecific defect can be created (8), may be equivalent to the anticodon arm of tRNA (6). Of these three domains, domain 3 is known to interact with eRF3 (9)(10)(11).…”

Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome

“…Oligoribonucleotides used as mRNAs were from Sigma-Aldrich. Generation of DNA constructs containing sequences coding for human eRF1 (full length) and eRF3 (lacking the nonessential amino-terminal 138 amino acids) and the preparation and purification of the respective recombinant proteins were carried out as described (Frolova et al 1998(Frolova et al , 2000; measurement of the factor's activity was performed according to Frolova et al (1994Frolova et al ( , 1996.…”

Recruitment of a Ribosomal Release Factor for Light- and Stress-Dependent Regulation of petB Transcript Stability in Arabidopsis Chloroplasts

“…The intron positions in AtPrfB2 and Os-PrfB2 encoding the mitochondrial proteins are highly conserved but differ from those found in At-PrfB3 and OsPrfB3, strongly suggesting that At-PrfB3 directly originated from a duplication of At-PrfB1 before the divergence of monocots and dicots. Remarkably, PrfB3 neither contains the otherwise conserved tripeptide anticodon SPF, which determines release factor specificity in vivo in PrfB proteins Nakamura et al, 2000), nor the universally conserved GGQ motif, which is essential for the hydrolytic activity and represents a structural counterpart on the CCA-39 acceptor stem of the tRNA-aminoacyl group (Frolova et al, 2000) (Figure 1). Moreover, the corresponding nucleotides of both motifs were not simply replaced but rather cut out from the genes by deletion events (Figure 1; see Supplemental Figure 1 online).…”