The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli

Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNAfMet requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNAfMet. Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNAfMet, IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNAfMet, which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNAfMet induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation

Archaic chaos: intrinsically disordered proteins in Archaea

Background: Many proteins or their regions known as intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) lack unique 3D structure in their native states under physiological conditions yet fulfill key biological functions. Earlier bioinformatics studies showed that IDPs and IDRs are highly abundant in different proteomes and carry out mostly regulatory functions related to molecular recognition and signal transduction. Archaea belong to an intriguing domain of life whose members, being microbes, are characterized by a unique mosaic-like combination of bacterial and eukaryotic properties and include inhabitants of some of the most extreme environments on the planet. With the expansion of the archaea genome data (more than fifty archaea species from five different phyla are known now), and with recent improvements in the accuracy of intrinsic disorder prediction, it is time to re-examine the abundance of IDPs and IDRs in the archaea domain.Results: The abundance of IDPs and IDRs in 53 archaea species is analyzed. The amino acid composition profiles of these species are generally quite different from each other. The disordered content is highly species-dependent. Thermoproteales proteomes have 14% of disordered residues, while in Halobacteria, this value increases to 34%. In proteomes of these two phyla, proteins containing long disordered regions account for 12% and 46%, whereas 4% and 26% their proteins are wholly disordered. These three measures of disorder content are linearly correlated with each other at the genome level. There is a weak correlation between the environmental factors (such as salinity, pH and temperature of the habitats) and the abundance of intrinsic disorder in Archaea, with various environmental factors possessing different disorder-promoting strengths. Harsh environmental conditions, especially those combining several hostile factors, clearly favor increased disorder content. Intrinsic disorder is highly abundant in functional Pfam domains of the archaea origin. The analysis based on the disordered content and phylogenetic tree indicated diverse evolution of intrinsic disorder among various classes and species of Archaea.Conclusions: Archaea proteins are rich in intrinsic disorder. Some of these IDPs and IDRs likely evolve to help archaea to accommodate to their hostile habitats. Other archaean IDPs and IDRs possess crucial biological functions similar to those of the bacterial and eukaryotic IDPs/IDRs

Conformational transitions in the gamma subunit of the archaeal translation initiation factor 2

In eukaryotes and archaea, the heterotrimeric translation initiation factor 2 e aIF2 is pivotal for the delivery of methionylated initiator tRNA Met tRNAi to the ribosome. It acts as a molecular switch that cycles between inactive GDP bound and active GTP bound states. Recent studies show that eIF2 can also exist in a long lived eIF2[gamma] GDP Pi inorganic phosphate active state. Here, four high resolution crystal structures of aIF2[gamma] from Sulfolobus solfataricus are reported aIF2[gamma] GDPCP a nonhydrolyzable GTP analogue , aIF2[gamma] GDP formate in which a formate ion possibly mimics Pi , aIF2[gamma] GDP and nucleotide free aIF2[gamma]. The structures describe the different states of aIF2[gamma] and demonstrate the conformational transitions that take place in the aIF2[gamma] life cycl

The third structural switch in the archaeal translation initiation factor 2 aIF2 molecule and its possible role in the initiation of GTP hydrolysis and the removal of aIF2 from the ribosome

The structure of the amp; 947; subunit of archaeal translation initiation factor 2 aIF2 from Sulfolobus solfataricus SsoIF2 amp; 947; 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 non crystallographic twofold symmetry axis, while the fifth has no symmetry equivalent. Analysis of the structure and its comparison with other known aIF2 amp; 947; 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 SsoIF2 amp; 947; 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 recognitio

Crystal Structure of the Human Ribosome in Complex with DENR-MCT-1

The repertoire of the density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein was recently expanded to include translational control of a specific set of cancer-related mRNAs. DENR and MCT-1 form the heterodimer, which binds to the ribosome and operates at both translation initiation and reinitiation steps, though by a mechanism that is yet unclear. Here, we determined the crystal structure of the human small ribosomal subunit in complex with DENR-MCT-1. The structure reveals the location of the DENR-MCT-1 dimer bound to the small ribosomal subunit. The binding site of the C-terminal domain of DENR on the ribosome has a striking similarity with those of canonical initiation factor 1 (eIF1), which controls the fidelity of translation initiation and scanning. Our findings elucidate how the DENR-MCT-1 dimer interacts with the ribosome and have functional implications for the mechanism of unconventional translation initiation and reinitiation

The effect of electric potential on the fracture surface of aluminum under creep tests

В данной работе методом просвечивающей электронной микроскопии выполнен анализ поверхности разрушения технически чистого алюминия, деформированного при ползучести в условиях приложения электрических потенциалов 1 В, 5 В и без электрического воздействия. В ходе исследований был выявлен негативный фактор влияния электрического потенциала на процесс ползучести, приводящий к увеличению скорости ползучести на установившейся стадии и снижению долговечности материала. Установлено, что подведение электрического потенциала к поверхности образцов технически чистого алюминия, подвергаемых испытаниям на ползучесть, отражается на изменении количественных параметров, характеризующих структуру разрушения. Показано, что приложение электрических потенциалов до 5 В сопровождается формированием широкого диапазона размеров ямок сдвига и, соответственно, снижением вязкости разрушения при испытаниях на ползучесть, а также уменьшением длительности испытаний, что может привести к ускоренному разрушению изделий, эксплуатирующихся в условиях влияния электрического потенциала.In the present study fracture surface analysis was performed under creep tests of pure aluminum with applied electric potential of 1V, 5V, as well as without electrical action. For this investigation a scanning electron microscopy method was used. In research a negative effect of the electric potential on creep performance resulting in a creep rate increase and material durability reduction has been discovered. It has been found out that application of electic potential to the surface of the samples of pure aluminum under creep test leads to quantitative changes of the parameters the fracture structure is characterized by. It is demonstrated that application of electric potential up to 5 V is accompanied by pits of fracture of wide range of sizes and consequently by fracture viscosity reduction upon creep testing, as well as test time reduction. All this can cause premature failure of the item in service when electric potential is applied

Cryo-EM study of start codon selection during archaeal translation initiation

Eukaryotic and archaeal translation initiation complexes have a common structural core comprising e/aIF1, e/aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNA(i)(Met)) and mRNA bound to the small ribosomal subunit. e/aIF2 plays a crucial role in this process but how this factor controls start codon selection remains unclear. Here, we present cryo-EM structures of the full archaeal 30S initiation complex showing two conformational states of the TC. In the first state, the TC is bound to the ribosome in a relaxed conformation with the tRNA oriented out of the P site. In the second state, the tRNA is accommodated within the peptidyl (P) site and the TC becomes constrained. This constraint is compensated by codon/anticodon base pairing, whereas in the absence of a start codon, aIF2 contributes to swing out the tRNA. This spring force concept highlights a mechanism of codon/anticodon probing by the initiator tRNA directly assisted by aIF2

Structure of the ternary initiation complex aIF2-GDPNP-methionylated initiator tRNA.

International audienceEukaryotic and archaeal translation initiation factor 2 (e/aIF2) is a heterotrimeric GTPase that has a crucial role in the selection of the correct start codon on messenger RNA. We report the 5-Å resolution crystal structure of the ternary complex formed by archaeal aIF2 from Sulfolobus solfataricus, the GTP analog GDPNP and methionylated initiator tRNA. The 3D model is further supported by solution studies using small-angle X-ray scattering. The tRNA is bound by the α and γ subunits of aIF2. Contacts involve the elbow of the tRNA and the minor groove of the acceptor stem, but not the T-stem minor groove. We conclude that despite considerable structural homology between the core γ subunit of aIF2 and the elongation factor EF1A, these two G proteins of the translation apparatus use very different tRNA-binding strategies