ABSTRACT
Eukaryotic and archaeal translation initiation factor 2 in complex with GTP delivers the initiator methionyl-tRNA to the small ribosomal subunit. Over the past 20 years, thanks to the efforts of various research groups, including ours, this factor from the archaeon Sulfolobus solfataricus and its individual subunits have been crystallized in ten different space groups. Analysis of the molecular packing in these crystals makes it possible to better understand the roles of functionally significant switches and other elements of the nucleotide-binding pocket during the function of the factor as well as the influence of external effects on its transition between active and inactive states.
Subject(s)
Archaeal Proteins , Sulfolobus solfataricus , Sulfolobus solfataricus/chemistry , Sulfolobus solfataricus/metabolism , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Crystallography, X-Ray , Models, Molecular , Guanosine Triphosphate/metabolism , Guanosine Triphosphate/chemistry , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/metabolism , Protein Conformation , Binding Sites , RNA, Transfer, Met/chemistry , RNA, Transfer, Met/metabolismABSTRACT
A full analysis has been conducted of the sequences and secondary structures of viral type-I or related IRESs identified in all of the elements that correspond to the previously described minimal fragment of the enterovirus C IRES, which mimics the glycine tRNA anticodon hairpin in the IRES structure and is necessary for the specific binding of glycyl-tRNA synthetase. Experiments on human glycyl-tRNA synthetase binding with the mRNA fragments of several taxonomically distant viruses showed that the binding constants of these complexes are similar. These results indicate that the regulation of translation initiation via glycyl-tRNA synthetase must be a universal mechanism for these viruses and the corresponding parts of their mRNAs must have similar spatial structures. Furthermore, at least one additional mRNA hairpin with the glycyl anticodon loop has been found in all analyzed viral type-I IRESs. It seems plausible that this extra hairpin is associated with the second RNA-binding site of the glycyl-tRNA synthetase dimer and stabilizes its complex with the viral mRNA.
Subject(s)
Glycine-tRNA Ligase/metabolism , Internal Ribosome Entry Sites , Peptide Chain Initiation, Translational , Humans , RNA, Messenger/genetics , RNA, Viral/genetics , RNA-Binding Proteins/metabolismABSTRACT
The currently available structural information is insufficient for a detailed analysis of interactions between human glycyl-tRNA synthetase (GARS) and enterovirus IRESs. At the same time, this information is required in order to understand how this IRES trans-acting factor (ITAF) functions during viral mRNA translation, which is in turn crucial for the development of direct-action antiviral agents. In this paper, a theoretical model of the complex between a cadicivirus A IRES fragment and the anticodon-binding domain of human GARS is constructed using molecular dynamics simulation based on all of the available structural and biochemical data. The proposed model enables the structural interpretation of the previously obtained biochemical data.
Subject(s)
Anticodon/chemistry , Glycine-tRNA Ligase/chemistry , Internal Ribosome Entry Sites , Humans , Models, Molecular , Peptide Chain Initiation, TranslationalABSTRACT
The genus Enterovirus combines a portion of small (+)ssRNA-containing viruses and is divided into 10 species of true enteroviruses and three species of rhinoviruses. These viruses are causative agents of the widest spectrum of severe and deadly epidemic diseases of higher vertebrates, including humans. Their ubiquitous distribution and high pathogenicity motivate active search to counteract enterovirus infections. There are no sufficiently effective drugs targeted against enteroviral diseases, thus treatment is reduced to supportive and symptomatic measures. This makes it extremely urgent to develop drugs that directly affect enteroviruses and hinder their development and spread in infected organisms. In this review, we cover the classification of enteroviruses, mention the most common enterovirus infections and their clinical manifestations, and consider the current state of development of anti-enteroviral drugs. One of the most promising targets for such antiviral drugs is the viral Internal Ribosome Entry Site (IRES). The classification of these elements of the viral mRNA translation system is also examined.
Subject(s)
Antiviral Agents/pharmacology , Enterovirus Infections/drug therapy , Enterovirus/classification , Enterovirus/pathogenicity , Enterovirus Infections/diagnosis , Humans , Internal Ribosome Entry Sites/drug effectsABSTRACT
The crystal structure of the γ-subunit of translation initiation factor 2 from the archaeon Sulfolobus solfataricus (SsoIF2γ) has been solved based on perfectly hemihedral twinned data. The protein was cocrystallized with the 10-fold molar excess of GTP analog (GDPCP) over protein. However, no nucleotide was found in the structure, and the model demonstrated the apo form of the protein. Two slightly different molecules in the asymmetric unit of the crystal are related by the non-crystallographic 2-fold axis and form a tightly associated dimer. This dimer is stabilized by an intermolecular hydrophobic core and hydrogen bonds. Lack of GDPCP in the nucleotide-binding pocket of the γ-subunit and significant excess of dimers over monomers in the crystallization solution suggest that these dimers are the building blocks of the crystal. Contrary to SsoIF2γ monomers, these dimers are able to crystallize in two oppositely oriented slightly different crystal domains, thus forming a twinned crystal. Comparison of crystallization conditions for the twinned and untwinned crystals of apo SsoIF2γ showed that stabilization of the dimers in the solution may be caused by higher sodium salt concentration. Since amino acid residues involved in intermolecular contacts in the dimer are responsible for binding of the γ- and α-subunits within SsoIF2, increase in sodium salt concentration may prevent functioning of SsoIF2 in the cell.
Subject(s)
Peptide Initiation Factors/chemistry , Protein Subunits/chemistry , Sulfolobus solfataricus/chemistry , Crystallography, X-RayABSTRACT
Aminoacyl-tRNA synthetases are an ancient enzyme family that specifically charge a tRNA molecule with a cognate amino acid required for protein synthesis. Glycyl-tRNA synthetase is one of the most interesting aminoacyl-tRNA synthetases due to its structure variability and functional features in the different organisms. It was shown recently that human glycyl-tRNA synthetase is a regulator of translational initiation of poliovirus mRNA. Details of this process and its mechanism still remain unknown. While exploring this stage of poliovirus functioning we have studied the interaction of the cytoplasmic form of human glycyl-tRNA synthetase and its domains with the fragments of the poliovirus IRES element. As a result, we have identified the minimal fragment of viral mRNA with which glycyl-tRNA synthetase fully interacts and estimated the contribution of some domains to the interaction of glycyl-tRNA synthetase with RNA.
Subject(s)
Glycine-tRNA Ligase/chemistry , RNA, Messenger/chemistry , RNA, Transfer/chemistry , Amino Acids/chemistry , Cytoplasm/chemistry , Glycine-tRNA Ligase/genetics , Humans , Poliovirus/chemistry , Poliovirus/enzymology , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Transfer/geneticsABSTRACT
An acute case of septic typhoid listeriosis revealed during diagnostic examination is reported
Subject(s)
Listeria/pathogenicity , Listeriosis/diagnosis , Sepsis/diagnosis , Child , Diagnosis, Differential , Female , Humans , Listeria/isolation & purification , Listeriosis/microbiology , Listeriosis/physiopathology , Sepsis/microbiology , Sepsis/physiopathologyABSTRACT
The structure of the intact heterotrimeric translation initiation factor 2 (e/aIF2) is of great interest due to its key role in the initiator tRNA delivery to the ribosome and in translation initiation regulation in eukaryotes and archaea. We have chosen aIF2 from the hyperthermophilic archaeobacterium Sulfolobus solfataricus (SsoIF2) as an object for crystallization and structural investigations. Genes of the SsoIF2 subunits alpha, beta, and gamma were cloned and superexpressed. A method for heterotrimer SsoIF2alphabetagamma purification was elaborated with at least 95% purity. Highly ordered crystals of the full-sized SsoIF2, reflecting X-rays at the resolution up to 2.8 A, were obtained for the first time.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/isolation & purification , Prokaryotic Initiation Factor-2/chemistry , Prokaryotic Initiation Factor-2/isolation & purification , Sulfolobus solfataricus/chemistry , Archaeal Proteins/genetics , Crystallization , Prokaryotic Initiation Factor-2/genetics , Protein Subunits/chemistry , Protein Subunits/isolation & purification , Recombinant Proteins/isolation & purificationABSTRACT
Nine mutant forms of ribosomal proteins L1 from the bacterium Thermus thermophilus and the archaeon Methanococcus jannaschii were obtained. Their crystal structures were determined and analyzed. Earlier determined structure of S179C TthL1 was also thoroughly analyzed. Five from ten mutant proteins reveal essential changes of spatial structure caused by surface point mutation. It proves that for correct studies of biological processes by site-directed mutagenesis it is necessary to determine or at least to model spatial structures of mutant proteins. Detailed comparison of mutant L1 structures with that of corresponding wild type proteins reveals that side chain of a mutated amino acid residue tries to locate like the side chain of the original residue in the wild type protein. This observation helps to model the mutant structures.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Amino Acid Sequence , Crystallography, X-Ray , Methanococcus/metabolism , Molecular Sequence Data , Mutation , Protein Conformation , Thermus thermophilus/metabolismABSTRACT
Crystal structures of unbound protein L1 and of its complexes with ribosomal an messenger RNAs are analyzed. It is shown that the values of the apparent association rate constant for L1-RNA depend on conformation of unbound protein L1. It is suggested that L1 binds to rRNA with higher affinity than to mRNA because of additional interactions between domain II of L1 and the loop rRNA region, which is absent in mRNA.
Subject(s)
RNA, Messenger/metabolism , RNA, Ribosomal/metabolism , Ribosomal Proteins/metabolism , Archaeal Proteins/metabolism , Bacterial Proteins/metabolism , Models, Molecular , Protein Binding , Protein Biosynthesis , Protein Conformation , RNA, Archaeal/metabolism , RNA, Bacterial/metabolismABSTRACT
Properties of specific interaction between ribosomal proteins and ribosomal RNAs were analyzed and a method for determination of "recognizing modules" on the protein surface was proposed. The method is based on the search of protein atoms making conserved H-bonds with RNA and forming an invariant spatial structure in homologous rRNA-protein complexes and in the isolated protein. A potential of the method is demonstrated on the determination of the recognizing modules on the surfaces of ribosomal proteins S8, S15 and L5.
Subject(s)
RNA, Ribosomal/metabolism , Ribosomal Proteins/physiology , Bacteria/metabolism , Hydrogen Bonding , Molecular Structure , Mutation/genetics , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolismABSTRACT
The domain structure of an antiferromagnetic superlattice is studied. Synchrotron Mössbauer and polarized neutron reflectometric maps show micrometer-size primary domain formation as the external field decreases from saturation to remanence. A secondary domain state consisting mainly of at least 1 order of magnitude larger domains is created when a small field along the layer magnetizations induces a bulk-spin-flop transition. The domain-size distribution is reproducibly dependent on the magnetic prehistory. The condition for domain coarsening is shown to be the equilibrium of the external field energy with the anisotropy energy.
ABSTRACT
The crystal structure of ribosomal protein S8 bound to its target 16 S rRNA from a hyperthermophilic archaeon Methanococcus jannaschii has been determined at 2.6 A resolution. The protein interacts with the minor groove of helix H21 at two sites located one helical turn apart, with S8 forming a bridge over the RNA major groove. The specificity of binding is essentially provided by the C-terminal domain of S8 and the highly conserved nucleotide core, characterized by two dinucleotide platforms, facing each other. The first platform (A595-A596), which is the less phylogenetically and structurally constrained, does not directly contact the protein but has an important shaping role in inducing cross-strand stacking interactions. The second platform (U641-A642) is specifically recognized by the protein. The universally conserved A642 plays a pivotal role by ensuring the cohesion of the complex organization of the core through an array of hydrogen bonds, including the G597-C643-U641 base triple. In addition, A642 provides the unique base-specific interaction with the conserved Ser105, while the Thr106 - Thr107 peptide link is stacked on its purine ring. Noteworthy, the specific recognition of this tripeptide (Thr-Ser-Thr/Ser) is parallel to the recognition of an RNA tetraloop by a dinucleotide platform in the P4-P6 ribozyme domain of group I intron. This suggests a general dual role of dinucleotide platforms in recognition of RNA or peptide motifs. One prominent feature is that conserved side-chain amino acids, as well as conserved bases, are essentially involved in maintaining tertiary folds. The specificity of binding is mainly driven by shape complementarity, which is increased by the hydrophobic part of side-chains. The remarkable similarity of this complex with its homologue in the T. thermophilus 30 S subunit indicates a conserved interaction mode between Archaea and Bacteria.
