The heterotrimeric (αβγ) translation initiation factor 2 of archaea and eukaryotes (a/eIF2) supplies the P-site of the ribosome with the initiation tRNA. Its two subunits (β 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 aIF2β 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 β- and γ-subunits of the aIF2 are different.
Eukaryotic and archaeal translation initiation factor 2 (e/aIF2) functions as a heterotrimeric complex. It consists of three subunits (α, β, γ). α- and β-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 βγ complexes is necessary for its proper functioning. In this work, we introduced mutations in the recognition part of the βγ 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 β-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 β-subunit, which stabilizes helical conformation of the switch.
The human YB-1 protein plays multiple cellular roles, of which many are dictated by its binding to RNA and DNA through its Cold Shock Domain (CSD). Using molecular dynamics simulation approaches ...validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA. The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP), albeit some differences in sequence specificity. The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.
Ribosomal stalk is involved in the formation of the so-called “GTPase-associated site” and plays a key role in the interaction of ribosome with translation factors and in the control of translation ...accuracy. The stalk is formed by two or three copies of the L7/L12 dimer bound to the C-terminal tail of protein L10. The N-terminal domain of L10 binds to a segment of domain II of 23S rRNA near the binding site for ribosomal protein L11. The structure of bacterial L10 in complex with three L7/L12 N-terminal dimers has been determined in the isolated state, and the structure of the first third of archaeal L10 bound to domain II of 23S rRNA has been solved within the Haloarcula marismortui 50S ribosomal subunit. A close structural similarity between the RNA-binding domain of archaeal L10 and the RNA-binding domain of bacterial L10 has been demonstrated. In this work, a long RNA-binding N-terminal fragment of L10 from Methanococcus jannaschii has been isolated and crystallized. The crystal structure of this fragment (which encompasses two-thirds of the protein) has been solved at 1.6 Å resolution. The model presented shows the structure of the RNA-binding domain and the structure of the adjacent domain that exist in archaeal L10 and eukaryotic P0 proteins only. Furthermore, our model incorporated into the structure of the H. marismortui 50S ribosomal subunit allows clarification of the structure of the archaeal ribosomal stalk base.
The crystal structure of the 92-nucleotide L1-specific fragment of 23S rRNA from Haloarcula marismortui (Hma) has been determined at 3.3 Å resolution. Similar to the corresponding bacterial rRNA ...fragments, this structure contains joined helix 76-77 topped by an approximately globular structure formed by the residual part of the L1 stalk rRNA. The position of HmaL1 relative to the rRNA was found by its docking to the rRNA fragment using the L1-rRNA complex from Thermus thermophilus as a guide model. In spite of the anomalous negative charge of the halophilic archaeal protein, the conformation of the HmaL1-rRNA interface appeared to be very close to that observed in all known L1-rRNA complexes. The designed structure of the L1 stalk was incorporated into the H. marismortui 50S ribosomal subunit. Comparison of relative positions of L1 stalks in 50S subunits from H. marismortui and T. thermophilus made it possible to reveal the site of inflection of rRNA during the ribosome function.
A new chimeric protein, named WT-CIIA, was designed by connecting the proline-rich decapeptide PPPVPPYSAG to the C-terminus of the alpha-spectrin SH3 domain through a natural twelve-residue linker to ...obtain a single-chain model that would imitate intramolecular SH3-ligand interaction. The crystal structure of this fusion protein was determined at 1.7 Å resolution. The asymmetric unit of the crystal contained two SH3 globules contacting with one PPPVPPY fragment located between them. The domains are related by the twofold non-crystallographic axis and the ligand lies in two opposite orientations with respect to the conservative binding sites of SH3 domains.
The L1 protuberance of the 50S ribosomal subunit is implicated in the release/disposal of deacylated tRNA from the E site. The apparent mobility of this ribosomal region has thus far prevented an ...accurate determination of its three-dimensional structure within either the 50S subunit or the 70S ribosome. Here we report the crystal structure at 2.65 A resolution of ribosomal protein L1 from Sulfolobus acidocaldarius in complex with a specific 55-nucleotide fragment of 23S rRNA from Thermus thermophilus. This structure fills a major gap in current models of the 50S ribosomal subunit. The conformations of L1 and of the rRNA fragment differ dramatically from those within the crystallographic model of the T. thermophilus 70S ribosome. Incorporation of the L1-rRNA complex into the structural models of the T. thermophilus 70S ribosome and the Deinococcus radiodurans 50S subunit gives a reliable representation of most of the L1 protuberance within the ribosome.
The structure of the large ribosomal subunit from the halophilic archaeon Haloarcula marismortui (Hma) is the only crystal structure of an archaeal ribosomal particle that has been determined to ...date. However, the first model of the Hma 50S ribosomal subunit contained some gaps: the structures of functionally important mobile lateral protuberances were not visualized. Subsequently, some parts of the P (L12) stalk base were visualized at 3.0 Å resolution Kavran & Steitz (2007), J. Mol. Biol.371, 1047–1059: the RNA‐binding domain of r‐protein P0 (L10), the C‐terminal domain of L11 and helices 43 and 44 of the 23 S rRNA. Here, the 2.4 Å resolution electron‐density map of the Hma 50S ribosomal subunit was revisited and approximately two‐thirds of the P0 protein, residues 1–58 of the N‐terminal domains of two P1 protein molecules, residues 130–156 of L11, the full‐length r‐protein LX, nucleotides 2137–2149 and 2226–2237 of the 23S rRNA helix H76 forming the L1 stalk, nucleotides 2339–2343 of the 23S rRNA (contacting L5 protein) and loops 29–34 and 108–128 of protein L5 could be visualized. Thus, this paper provides a supplemented version of the Hma 50S ribosomal subunit model.
The heterotrimeric (alphabetagamma) translation initiation factor 2 of archaea and eukaryotes (a/eIF2) supplies the P-site of the ribosome with the initiation tRNA. Its two subunits (beta and gamma) ...contain the Cys2âCys2 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 Cys2âCys2 motifs in the aIF2beta and aIF2gamma 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 beta- and gamma-subunits of the aIF2 are different.
The structure of the γ subunit of archaeal translation initiation factor 2 (aIF2) from Sulfolobus solfataricus (SsoIF2γ) was determined in complex with GDPCP (a GTP analog). Crystals were obtained in ...the absence of magnesium ions in the crystallization solution. They belonged to space group P1, with five molecules in the unit cell. Four of these molecules are related in pairs by a common noncrystallographic twofold symmetry axis, while the fifth has no symmetry equivalent. Analysis of the structure and its comparison with other known aIF2 γ‐subunit structures in the GTP‐bound state show that (i) the magnesium ion is necessary for the formation and the maintenance of the active form of SsoIF2γ and (ii) in addition to the two previously known structural switches 1 and 2, eukaryotic translation initiation factor 2 (eIF2) and aIF2 molecules have another flexible region (switch 3), the function of which may consist of initiation of the hydrolysis of GTP and the removal of e/aIF2 from the ribosome after codon–anticodon recognition.
The structure of the γ subunit of aIF2 (aIF2γ) in complex with GTPCP obtained in the absence of Mg2+ ions in the crystallization solution was determined and new information about the role of Mg2+ ions in the function of aIF2γ was provided. A new functional region (switch 3) of aIF2γ was discovered.
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