Abstract
Background:We have previously demonstrated that the eukaryote-specific ribosomal protein eL42 of the human 80S ribosome contains seven monomethylated residues, among which are the Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence conserved in all eukaryotic 80S ribosomes. This sequence contains the methylated and universally conserved GGQ motif common for all class-1 translation termination factors responsible for stop codon recognition and for triggering the hydrolysis of the P site-bound peptidyl-tRNA. We have also recently reported a model of ribosomal ternary eL42-tRNA-eRF1 complex where specific regions of all three macromolecules (the comparably flexible GGQ domains of eRF1 and eL42 and the CCA-arm of tRNA) are involved in interactions.Method:Here, we have studied the interactions between recombinant eL42 and eRF1 proteins and the tRNA substrate by means of the Biacore assay, using the wild-type eL42 protein, the eL42-Δ(GGQTK) mutant (the eL42 protein whose GGQTK motif has been deleted), the single Q51E and K53Q mutants (eL42-Q51E and eL42-K53Q, respectively), as well as the double Q51A/K53A mutant (eL42-Q51A/K53A).Results:Our results show that the monomethylated Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence of eL42 and the monomethylated GGQ motif of eRF1 represents the sites of interaction between these two proteins through hydrophobic contacts between methyl groups. We also demonstrate that the interactions between eL42 and tRNA or 28S rRNA are characterized by strong binding affinities (KD values in the nanomolar or picomolar range, respectively) which argue for specific interactions. Strong interactions between eL42 and tRNA are likely to be responsible for the decrease in the poly(U)-dependent poly(Phe) synthesis activity of human 80S or E. coli 70S ribosomes in the presence of added human recombinant eL42. It is proposed that the decrease of the activity of the ribosome is caused by the sequestration of the substrate Phe-tRNAPhe by the added eL42 protein.Conclusion:Interactions between the monomethylated Gln-51 and Lys-53 residues of the 49GGQTK53 motif of the human eL42 protein and the methylated GGQ motif of eRF1 are likely to play a functional role on translating human 80S ribosomes.
Highlights
Protein methylation, the transfer of a methyl group to amino acids, such as lysines, arginines and histidines, is an essential process for the regulation of gene expression, protein function, and RNA metabolism [1, 2]
Our results show that the methylated residues of the GGQ minidomains of the human proteins eL42 and eRF1 represent the sites of interaction between these two proteins through hydrophobic contacts between methyl groups
Effects of added recombinant eL42 protein to the activity of human 80S ribosomes. Another question that we have addressed in the present report is the effect of added eL42 protein on the activity of human 80S ribosomes. This question stemmed from the following observations: (i) with the combination of biochemical and genetical approaches, we have recently demonstrated that the ribosomal protein eL42 from eukaryotes is indispensable for the activity of the yeast 80S ribosomes; (ii) the human eL42 protein was previously shown to be overexpressed in human hepatocellular carcinoma (HCC) as well as in several human tumor cell-lines [31, 32]
Summary
The transfer of a methyl group to amino acids, such as lysines, arginines and histidines, is an essential process for the regulation of gene expression, protein function, and RNA metabolism [1, 2]. Our recent studies have shown that the eukaryote-specific large subunit ribosomal protein eL42 of the human 80S ribosome contains seven monomethylated residues, among which are the Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence conserved in all eukaryotic 80S ribosomes [9] This sequence contains the methylated and universally conserved GGQ motif common for all class-1 translation termination factors responsible for stop codon recognition and for triggering the hydrolysis of the P site-bound peptidyl-tRNA [10 - 13]. We have previously demonstrated that the eukaryote-specific ribosomal protein eL42 of the human 80S ribosome contains seven monomethylated residues, among which are the Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence conserved in all eukaryotic 80S ribosomes This sequence contains the methylated and universally conserved GGQ motif common for all class-1 translation termination factors responsible for stop codon recognition and for triggering the hydrolysis of the P site-bound peptidyltRNA. We have recently reported a model of ribosomal ternary eL42-tRNA-eRF1 complex where specific regions of all three macromolecules (the comparably flexible GGQ domains of eRF1 and eL42 and the CCA-arm of tRNA) are involved in interactions
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