Phosphorylation of more than one site is required for tight interaction of human tau protein with 14-3-3ζ

AbstractSerine residues phosphorylated by protein kinase A (PKA) in the shortest isoform of human tau protein (τ3) were sequentially replaced by alanine and interaction of phosphorylated τ3 and its mutants with 14-3-3 was investigated. Mutation S156A slightly decreased interaction of phosphorylated τ3 with 14-3-3. Double mutations S156A/S267A and especially S156A/S235A, strongly inhibited interaction of phosphorylated τ3 with 14-3-3. Thus, two sites located in the Pro-rich region and in the pseudo repeats of τ3 are involved in phosphorylation-dependent interaction of τ3 with 14-3-3. The state of τ3 phosphorylation affects the mode of 14-3-3 binding and by this means might modify tau filament formation.Structured summaryMINT-7233358, MINT-7233372, MINT-7233384: 14-3-3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636-3) bind (MI:0407) by molecular sieving (MI:0071)MINT-7233323, MINT-7233334, MINT-7233346: Tau 3 (uniprotkb:P10636-3) and 14-3-3 zeta (uniprotkb:P63104) bind (MI:0407) by crosslinking studies (MI:0030)MINT-7233285, MINT-7233297, MINT-7233310: 14-3-3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636-3) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404

Invariant amino acids essential for decoding function of polypeptide release factor eRF1

In eukaryotic ribosome, the N domain of polypeptide release factor eRF1 is involved in decoding stop signals in mRNAs. However, structure of the decoding site remains obscure. Here, we specifically altered the stop codon recognition pattern of human eRF1 by point mutagenesis of the invariant Glu55 and Tyr125 residues in the N domain. The 3D structure of generated eRF1 mutants was not destabilized as demonstrated by calorimetric measurements and calculated free energy perturbations. In mutants, the UAG response was most profoundly and selectively affected. Surprisingly, Glu55Arg mutant completely retained its release activity. Substitution of the aromatic ring in position 125 reduced response toward all stop codons. This result demonstrates the critical importance of Tyr125 for maintenance of the intact structure of the eRF1 decoding site. The results also suggest that Tyr125 is implicated in recognition of the 3d stop codon position and probably forms an H-bond with Glu55. The data point to a pivotal role played by the YxCxxxF motif (positions 125–131) in purine discrimination of the stop codons. We speculate that eRF1 decoding site is formed by a 3D network of amino acids side chains

Molecular dissection of translation termination mechanism identifies two new critical regions in eRF1

Translation termination in eukaryotes is completed by two interacting factors eRF1 and eRF3. In Saccharomyces cerevisiae, these proteins are encoded by the genes SUP45 and SUP35, respectively. The eRF1 protein interacts directly with the stop codon at the ribosomal A-site, whereas eRF3—a GTPase protein—probably acts as a proofreading factor, coupling stop codon recognition to polypeptide chain release. We performed random PCR mutagenesis of SUP45 and screened the library for mutations resulting in increased eRF1 activity. These mutations led to the identification of two new pockets in domain 1 (P1 and P2) involved in the regulation of eRF1 activity. Furthermore, we identified novel mutations located in domains 2 and 3, which confer stop codon specificity to eRF1. Our findings are consistent with the model of a closed-active conformation of eRF1 and shed light on two new functional regions of the protein

A single amino acid change of translation termination factor eRF1 switches between bipotent and omnipotent stop-codon specificity†

In eukaryotes a single class-1 translation termination factor eRF1 decodes the three stop codons: UAA, UAG and UGA. Some ciliates, like Euplotes, have a variant code, and here eRF1s exhibit UAR-only specificity, whereas UGA is reassigned as a sense codon. Since eukaryote eRF1 stop-codon recognition is associated with its N-terminal domain, structural features should exist in the N domain of ciliate eRF1s that restrict their stop-codon specificity. Using an in vitro reconstituted eukaryotic translation system we demonstrate here that a chimeric eRF1 composed of the N domain of Euplotes aediculatus eRF1 fused to the MC domains of human eRF1 exhibits UAR-only specificity. Functional analysis of eRF1 chimeras constructed by swapping Euplotes N domain sequences with the cognate regions from human eRF1 as well as site-directed mutagenesis of human eRF1 highlighted the crucial role of the alanine residue in position 70 of E. aediculatus eRF1 in restricting UGA decoding. Switching the UAR-only specificity of E. aediculatus eRF1 to omnipotent mode is due to a single point mutation. Furthermore, we examined the influence of eRF3 on the ability of chimeric and mutant eRF1s to induce peptide release in response to different stop codons

New insights into stop codon recognition by eRF1

In eukaryotes, translation termination is performed by eRF1, which recognizes stop codons via its N-terminal domain. Many previous studies based on point mutagenesis, cross-linking experiments or eRF1 chimeras have investigated the mechanism by which the stop signal is decoded by eRF1. Conserved motifs, such as GTS and YxCxxxF, were found to be important for termination efficiency, but the recognition mechanism remains unclear. We characterized a region of the eRF1 N-terminal domain, the P1 pocket, that we had previously shown to be involved in termination efficiency. We performed alanine scanning mutagenesis of this region, and we quantified in vivo readthrough efficiency for each alanine mutant. We identified two residues, arginine 65 and lysine 109, as critical for recognition of the three stop codons. We also demonstrated a role for the serine 33 and serine 70 residues in UGA decoding in vivo. NMR analysis of the alanine mutants revealed that the correct conformation of this region was controlled by the YxCxxxF motif. By combining our genetic data with a structural analysis of eRF1 mutants, we were able to formulate a new model in which the stop codon interacts with eRF1 through the P1 pocket

Selectivity of stop codon recognition in translation termination is modulated by multiple conformations of GTS loop in eRF1

Translation termination in eukaryotes is catalyzed by two release factors eRF1 and eRF3 in a cooperative manner. The precise mechanism of stop codon discrimination by eRF1 remains obscure, hindering drug development targeting aberrations at translation termination. By solving the solution structures of the wild-type N-domain of human eRF1 exhibited omnipotent specificity, i.e. recognition of all three stop codons, and its unipotent mutant with UGA-only specificity, we found the conserved GTS loop adopting alternate conformations. We propose that structural variability in the GTS loop may underline the switching between omnipotency and unipotency of eRF1, implying the direct access of the GTS loop to the stop codon. To explore such feasibility, we positioned N-domain in a pre-termination ribosomal complex using the binding interface between N-domain and model RNA oligonucleotides mimicking Helix 44 of 18S rRNA. NMR analysis revealed that those duplex RNA containing 2-nt internal loops interact specifically with helix α1 of N-domain, and displace C-domain from a non-covalent complex of N-domain and C-domain, suggesting domain rearrangement in eRF1 that accompanies N-domain accommodation into the ribosomal A site