Oleg Nikonov

Ribosomal protein L1 recognizes the same specific structural motif in its target sites on the autoregulatory mRNA and 23S rRNA

The RNA-binding ability of ribosomal protein L1 is of profound interest since the protein has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding its mRNA. Here, we report the crystal structure of ribosomal protein L1 in complex with a specific fragment of its mRNA and compare it with the structure of L1 in complex with a specific fragment of 23S rRNA determined earlier. In both complexes, a strongly conserved RNA structural motif is involved in L1 binding through a conserved network of RNA–protein H-bonds inaccessible to the solvent. These interactions should be responsible for specific recognition between the protein and RNA. A large number of additional non-conserved RNA–protein H-bonds stabilizes both complexes. The added contribution of these non-conserved H-bonds makes the ribosomal complex much more stable than the regulatory one.

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.

Crystal Structure of the Archaeal Translation Initiation Factor 2 in Complex with a GTP Analogue and Met-tRNAfMet

Heterotrimeric aIF2αβγ (archaeal homologue of the eukaryotic translation initiation factor 2) in its GTP-bound form delivers Met-tRNAi(Met) to the small ribosomal subunit. It is known that the heterodimer containing the GTP-bound γ subunit and domain 3 of the α subunit of aIF2 is required for the formation of a stable complex with Met-tRNAi. Here, the crystal structure of an incomplete ternary complex including aIF2αD3γ⋅GDPNP⋅Met-tRNAf(Met) has been solved at 3.2Å resolution. This structure is in good agreement with biochemical and hydroxyl radical probing data. The analysis of the complex shows that despite the structural similarity of aIF2γ and the bacterial translation elongation factor EF-Tu, their modes of tRNA binding are very different. Remarkably, the recently published 5.0-Å-resolution structure of almost the same ternary initiation complex differs dramatically from the structure presented. Reasons for this discrepancy are discussed.

[Identification of RNA-recognizing modules on the surface of ribosomal proteins].

Specific binding of ribosomal proteins to rRNA has been analyzed, and the method for determining the recognizing modules on the protein surface has been proposed. This method is based on the search for the atoms on the protein molecule that are involved in the conserved hydrogen bonds with rRNA and form invariant spatial structure in both free and RNA-bound ribosomal proteins. The potential of this method is illustrated by determining the rRNA-recognizing modules on the surface of ribosomal proteins S8, S15, and L5.

Crystal structures of mutant ribosomal proteins L1

Nine mutant ribosomal proteins L1 from the bacterium Thermus thermophilus and archaeon Methanococcus jannaschii were obtained and their crystal structures were determined and analyzed. The structure of the S179C TthL1 mutant, determined earlier, was also analyzed. In half of the proteins studied, point mutations of the amino acid residues exposed on the protein surface essentially changed the spatial structure of the protein. This proves that a correct study of biological processes with the help of site-directed mutagenesis requires a preliminary determination or, at least, modeling of the structures of mutant proteins. A detailed comparison of the structures of the L1 mutants and the corresponding wild-type L1 proteins demonstrated that the side chain of a mutated amino acid residue tends to adopt a location similar to that of the side chain of the corresponding residue in the wild-type protein. This observation assists in modeling the structure of mutant proteins.

Crystallization of RNA/protein complexes

Different complexes of ribosomal proteins with specific rRNA fragments have been crystallized and studied by our group during the last six years. There are several factors important for successful crystallization of RNA/protein complexes, among them: length and content of RNA fragments, homogeneity of RNA and protein preparations, stability of the complexes, conditions for mixing RNA and protein components before crystallization, effect of Se-Met on RNA/protein complex crystal quality. In this paper we describe findings and methodical details, which helped us to succeed in obtaining X-ray quality crystals of several RNA/protein complexes.

Enteroviruses: Classification, diseases they cause, and approaches to development of antiviral drugs

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 pathogenici- ty 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 man- ifestations, 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.

Glycyl-tRNA Synthetase as a Potential Universal Regulator of Translation Initiation at IRES-I

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.

Crystallization of mutant forms of the γ subunit of archaeal translation initiation factor 2

Archaeal translation initiation factor 2 (aIF2) is homologous to its eukaryotic counterpart (eIF2). It is a heterotrimeric protein consisting of α, β, and γ subunits. The protein e/aIF2 forms a ternary complex with guanosine 5′-triphosphate and the initiator methionyl-tRNA (Met-tRNAi) and delivers the latter to the ribosome. In archaea, translation initiation factor 2 has an additional function. The γ subunit of aIF2 binds mRNAs with a triphosphate at the 5′-end and prevents 5′-to-3′ directional mRNA decay. To determine the mRNA-binding site on the surface of aIF2γ, mutations were introduced into the protein sequence at sites of possible interactions with mRNA. The crystals of the mutant forms of aIF2γ were obtained, and X-ray diffraction data sets suitable for structure determination at atomic resolution were collected.

Isolation and crystallization of heterotrimeric translation initiation factor 2 from Sulfolobus solfataricus

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 α, β, and γ were cloned and superexpressed. A method for heterotrimer SsoIF2αβγ 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 Å, were obtained for the first time.