Recognition of γ-Subunit by ß-Subunit in Translation Initiation Factor 2. Stabilization of the GTP-Bound State of I/F 2 in Archaea and Eukaryotes.

Eukaryotic and archaeal translation initiation factor 2 (e/aIF2) functions as a heterotrimeric complex. It consists of three subunits (α, ß, γ). α- and ß-subunits are bound to γ-subunit by hydrogen bonds and van der Waals interactions, but do not contact each other. Although main functions of the factor are performed by the γ-subunit, reliable formation of αγ and ßγ complexes is necessary for its proper functioning. In this work, we introduced mutations in the recognition part of the ßγ interface and showed that hydrophobic effect plays a crucial role in the recognition of subunits both in eukaryotes and archaea. Shape and properties of the groove on the surface of γ-subunit facilitates transition of the disordered recognition part of the ß-subunit into an α-helix containing approximately the same number of residues in archaea and eukaryotes. In addition, based on the newly obtained data, it was concluded that in archaea and eukaryotes, transition of the γ-subunit to the active state leads to additional contact between the region of switch 1 and C-terminal part of the ß-subunit, which stabilizes helical conformation of the switch.

VPg of Potato Virus Y and Potato Cap-Binding eIF4E Factors: Selective Interaction and Its Supposed Mechanism.

Potato virus Y (PVY) is one of the most common and harmful plant viruses. Translation of viral RNA starts with the interaction between the plant cap-binding translation initiation factors eIF4E and viral genome-linked protein (VPg) covalently attached to the viral RNA. Disruption of this interaction is one of the natural mechanisms of plant resistance to PVY. The multigene eIF4E family in the potato (Solanum tuberosum L.) genome contains genes for the translation initiation factors eIF4E1, eIF4E2, and eIF(iso)4E. However, which of these factors can be recruited by the PVY, as well as the mechanism of this interaction, remain obscure. Here, we showed that the most common VPg variant from the PVY strain NTN interacts with eIF4E1 and eIF4E2, but not with eIF(iso)4E. Based on the VPg, eIF4E1, and eIF4E2 models and data on the natural polymorphism of VPg amino acid sequence, we suggested that the key role in the recognition of potato cap-binding factors belongs to the R104 residue of VPg. To verify this hypothesis, we created VPg mutants with substitutions at position 104 and examined their ability to interact with potato eIF4E factors. The obtained data were used to build the theoretical model of the VPg-eIF4E2 complex that differs significantly from the earlier models of VPg complexes with eIF4E proteins, but is in a good agreement with the current biochemical data.

Structure and Function of Archaeal Translation Initiation Factor 2 Fragments Containing Cys2-Cys2 Motifs.

The heterotrimeric (αßγ) translation initiation factor 2 of archaea and eukaryotes (a/eIF2) supplies the P-site of the ribosome with the initiation tRNA. Its two subunits (ß and γ) contain the Cys2-Cys2 motif, which is capable of forming a stable zinc finger structure in the presence of zinc ions. In this work, comparative analysis of the fragments containing Cys2-Cys2 motifs in the aIF2ß and aIF2γ structures from different organisms was carried out and their environments in crystals was analyzed. Based on the obtained data, a conclusion was made that the conformation and role of these fragments in the ß- and γ-subunits of the aIF2 are different.

Enhanced C/EBP binding to G·T mismatches facilitates fixation of CpG mutations in cancer and adult stem cells.

Somatic mutations in regulatory sites of human stem cells affect cell identity or cause malignant transformation. By mining the human genome for co-occurrence of mutations and transcription factor binding sites, we show that C/EBP binding sites are strongly enriched with [C > T]G mutations in cancer and adult stem cells, which is of special interest because C/EBPs regulate cell fate and differentiation. In vitro protein-DNA binding assay and structural modeling of the CEBPB-DNA complex show that the G·T mismatch in the core CG dinucleotide strongly enhances affinity of the binding site. We conclude that enhanced binding of C/EBPs shields CpG·TpG mismatches from DNA repair, leading to selective accumulation of [C > T]G mutations and consequent deterioration of the binding sites. This mechanism of targeted mutagenesis highlights the effect of a mutational process on certain regulatory sites and reveals the molecular basis of putative regulatory alterations in stem cells.

Associations between Neurological Diseases and Mutations in the Human Glycyl-tRNA Synthetase.

Aminoacyl-RNA synthetases (aaRSs) are among the key enzymes of protein biosynthesis. They are responsible for conducting the first step in the protein biosynthesis, namely attaching amino acids to the corresponding tRNA molecules both in cytoplasm and mitochondria. More and more research demonstrates that mutations in the genes encoding aaRSs lead to the development of various neurodegenerative diseases, such as incurable Charcot-Marie-Tooth disease (CMT) and distal spinal muscular atrophy. Some mutations result in the loss of tRNA aminoacylation activity, while other mutants retain their classical enzyme activity. In the latter case, disease manifestations are associated with additional neuron-specific functions of aaRSs. At present, seven aaRSs (GlyRS, TyrRS, AlaRS, HisRS, TrpRS, MetRS, and LysRS) are known to be involved in the CMT etiology with glycyl-tRNA synthetase (GlyRS) being the most studied of them.

Multifaceted Mechanism of Amicoumacin A Inhibition of Bacterial Translation.

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami.

The crucial role of conserved intermolecular H-bonds inaccessible to the solvent in formation and stabilization of the TL5.5 SrRNA complex.

Analysis of the structures of two complexes of 5 S rRNA with homologous ribosomal proteins, Escherichia coli L25 and Thermus thermophilus TL5, revealed that amino acid residues interacting with RNA can be divided into two different groups. The first group consists of non-conserved residues, which form intermolecular hydrogen bonds accessible to solvent. The second group, comprised of strongly conserved residues, form intermolecular hydrogen bonds that are shielded from solvent. Site-directed mutagenesis was used to introduce mutations into the RNA-binding site of protein TL5. We found that replacement of residues of the first group does not influence the stability of the TL5.5 S rRNA complex, whereas replacement of residues of the second group leads to destabilization or disruption of the complex. Stereochemical analysis shows that the replacements of residues of the second group always create complexes with uncompensated losses of intermolecular hydrogen bonds. We suggest that these shielded intermolecular hydrogen bonds are responsible for the recognition between the protein and RNA.