[Regulation of Ribosomal Protein Synthesis in Prokaryotes].

Protein synthesis on ribosomes is considered the main process in cell life. Regulation of ribosomal protein gene expression plays an important role in the balanced synthesis of proteins and RNA in ribosomal biogenesis. This review is focused on some features of autoregulation of ribosomal protein synthesis in prokaryotes. Inhibition of the synthesis of ribosomal proteins encoded by 12 operons by mechanisms of competition , "entrapment", and retroregulation are discussed. Examples of regulation of protein synthesis by individual ribosomal proteins and their complexes are presented.

Crystal structures and RNA-binding properties of Lsm proteins from archaea Sulfolobus acidocaldarius and Methanococcus vannielii: Similarity and difference of the U-binding mode.

Sm and Sm-like (Lsm) proteins are considered as an evolutionary conserved family involved in RNA metabolism in organisms from bacteria and archaea to human. Currently, the function of Sm-like archaeal proteins (SmAP) is not well understood. Here, we report the crystal structures of SmAP proteins from Sulfolobus acidocaldarius and Methanococcus vannielii and a comparative analysis of their RNA-binding sites. Our data show that these SmAPs have only a uridine-specific RNA-binding site, unlike their bacterial homolog Hfq, which has three different RNA-binding sites. Moreover, variations in the amino acid composition of the U-binding sites of the two SmAPs lead to a difference in protein affinity for oligo(U) RNA. Surface plasmon resonance data and nucleotide-binding analysis confirm the high affinity of SmAPs for uridine nucleotides and oligo(U) RNA and the reduced affinity for adenines, guanines, cytidines and corresponding oligo-RNAs. In addition, we demonstrate that MvaSmAP1 and SacSmAP2 are capable of melting an RNA hairpin and, apparently, promote its interaction with complementary RNA.

Interleukin-17: Functional and Structural Features, Application as a Therapeutic Target.

Cytokines of the IL-17 family play a key role in the host organism defense against bacterial and fungal infections. At the same time, upregulated synthesis of IL-17 cytokines is associated with immunoinflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and others. The members of this family are important therapeutic targets in the treatment of various human chronic inflammatory disorders. Elucidation of signaling pathways involving IL-17 family proteins and analysis of the structure of cytokine complexes with specific antibodies, inhibitors, and receptors are essential for the development of new drugs for the therapy of immunoinflammatory rheumatic diseases.

Structural and functional properties of antimicrobial protein L5 of Lysоbacter sp. XL1.

The Gram-negative bacterium Lysobacter sp. XL1 secretes into the extracellular space five bacteriolytic enzymes that lyse the cell walls of competing microorganisms. Of special interest are homologous lytic proteases L1 and L5. This work found protein L5 to possess Gly-Gly endopeptidase and N-acetylmuramoyl-L-Ala amidase activities with respect to staphylococcal peptidoglycan. Protein L5 was found to be capable of aggregating into amyloid-like fibril structures. The crystal structure of protein L5 was determined at a 1.60-Å resolution. Protein L5 was shown to have a rather high structural identity with bacteriolytic protease L1 of Lysobacter sp. XL1 and α-lytic protease of Lysobacter enzymogenes at a rather low identity of their amino acid sequences. Still, the structure of protein L5 was revealed to have regions that differed from their equivalents in the homologs. The revealed structural distinctions in L5 are suggested to be of importance in exhibiting its unique properties.

[Incorporation of Copper Ions into T2/T3 Centers of Two-Domain Laccases].

Laccase belongs to the family of copper-containing oxidases. A study was made of the mechanism that sustains the incorporation of copper ions into the T2/T3 centers of recombinant two-domain laccase Streptomyces griseoflavus Ac-993. The occupancy of the T3 center by copper ions was found to increase with an increasing copper content in the culture medium and after dialysis of the protein preparation against a copper sulfate-containing buffer. The T2 center was filled only when overproducer strain cells were grown at a higher copper concentration in the medium. Two-domain laccases were assumed to possess a channel that serves to deliver copper ions to the T3 center during the formation of the three-dimensional laccase conformation and dialysis of the protein preparation. A narrower channel leads to the T2 center in two-domain laccases compared with three-domain ones, rendering the center less accessible for copper atoms. The incorporation of copper ions into the T2 center of two-domain laccases is likely to occur in the course of their biosynthesis or the formation of a functional trimer.

[Identification of Ribosomal Protein L1-Binding Sites in Thermus thermophilus and Thermotoga maritima mRNAs].

The conserved two-domain ribosomal protein (r-protein) L1 is a structural part of the L1 stalk of the large ribosomal subunit and regulates the translation of the operon that comprises its own gene. The regulatory properties of the bacterial r-protein L1 have only been studied in detail for Escherichia coli; however, there were no such studies for other bacteria, in particular, Thermus thermophilus and Thermotoga maritima, which are more evolutionarily ancient. It is known that domain I of the r-protein L1 might have regulatory properties of the whole protein. The aim of this study was to identify regulatory sites on the mRNA of T. thermophilus and T. maritima that interact with r-proteins L1, as well as with their domains I from the same organisms. An analysis of the mRNA of the L11 operon T. thermophilus showed the presence of one potential binding site of the L1 r-protein, two such regions were found also in the mRNA sequence of the L11 operon of T. maritima. The dissociation constants for the L1 proteins from T. thermophilus and T. maritima and their domains I with mRNA fragments from the same organisms that contain the supposed L1-binding sites were determined by surface plasmon resonance. It has been shown that the ribosomal proteins L1 as their domains I bind specific fragments of mRNA from the same organisms that may suggest regulatory activity of the L1 protein in the T. thermophilus and T. maritima and conservatism of the principles of L1-RNA interactions.

[Influence of Nonconserved Regions of L1 Protuberance of Thermus thermophilus Ribosome on the Affinity of L1 Protein to 23s rRNA].

The L1 protuberance of the ribosome includes two domain ribosomal protein L1 and three helices of 23S rRNA (H76, H77, and H78) with interconnecting loops A and B. Helix 78 consists of two parts, i.e., H78a and H78b. A comparison of the available structural data of L1-RNA complexes with the obtained kinetic data made it possible to determine the influence of the nonconserved regions of Thermus thermophilus L1-protuberance on the mutual affinity of the L1 protein and 23S rRNA. It has been shown that the N-terminal helix of the protein and 78b helix of 23S rRNA are essential for the formation of an additional intermolecular contact, which is separated in the protein from the main site of L1-rRNA interaction by a flexible connection. This results in a rise in the TthL1-rRNA affinity. At the same time, the elongation of the 76 helix has no effect on rRNA-protein binding.

Supramolecular organization of Hfq-like proteins.

Bacterial Hfq proteins are structural homologs of archaeal and eukaryotic Sm/Lsm proteins, which are characterized by a 5-stranded ß-sheet and an N-terminal α-helix. Previously, it was shown that archaeal Lsm proteins (SmAP) could produce long fibrils spontaneously, in contrast to the Hfq from Escherichia coli that could form similar fibrils only after special treatment. The organization of these fibrils is significantly different, but the reason for the dissimilarity has not been found. In the present work, we studied the process of fibril formation by bacterial protein Hfq from Pseudomonas aeruginosa and archaeal protein SmAP from Methanococcus jannaschii. Both proteins have high homology with E. coli Hfq. We found that Hfq from P. aeruginosa could form fibrils after substitutions in the conserved Sm2 motif only. SmAP from M. jannaschii, like other archaeal Lsm proteins, form fibrils spontaneously. Despite differences in the fibril formation conditions, the architecture of both was similar to that described for E. coli Hfq. Therefore, universal nature of fibril architecture formed by Hfq proteins is suggested.

Structural and functional characterization of two-domain laccase from Streptomyces viridochromogenes.

Laccase (EC 1.10.3.2) is one of the most common copper-containing oxidases found in many organisms and catalyses oxidation of primarily phenolic compounds by oxygen. A recently found bacterial laccase whose molecule is formed by two domains - the so called two-domain laccase (2DLac) or small laccase - has unusual resistance to inhibitors and an alkaline optimum of activity. The causes of these properties, as well as the biological function of two-domain laccases, are poorly understood. We performed an enzymatic and structural characterization of 2DLac from Streptomyces viridochromogenes (SvSL). It was cloned and overproduced in Escherichia coli. Phenolic compounds were oxidized in the presence of the enzyme under alkaline but not acidic conditions. Conversely, nonphenolic compounds were oxidized at acidic but not alkaline pH. SvSL catalysed oxidation of nonphenolic compounds more efficiently than that of phenols. Moreover, this two-domain laccase displayed a cytochrome c oxidase activity and exhibited no ferroxidase activity. The enzyme was resistant to specific inhibitors of copper-containing oxidases, such as NaN3 and NaF. We succeeded in generating X-ray quality crystals and solved their structure to a resolution of 2.4 Å. SvSL is a homotrimer in its native state. Comparison of its structure with that of a three-domain laccase revealed differences in the second coordination sphere of the T2/T3 centre and solvent channels. The role of these differences in the resistance of the enzyme to inhibitors and the activity at alkaline pH is under discussion.

Investigation of the regulatory function of archaeal ribosomal protein L4.

Ribosomal protein L4 is a regulator of protein synthesis in the Escherichia coli S10 operon, which contains genes of 11 ribosomal proteins. In this work, we have investigated regulatory functions of ribosomal protein L4 of the thermophilic archaea Methanococcus jannaschii. The S10-like operon from M. jannaschii encodes not 11, but only five ribosomal proteins (L3, L4, L23, L2, S19), and the first protein is L3 instead of S10. We have shown that MjaL4 and its mutant form lacking an elongated loop specifically inhibit expression of the first gene of the S10-like operon from the same organism in a coupled transcription-translation system in vitro. By deletion analysis, an L4-binding regulatory site has been found on MjaL3 mRNA, and a fragment of mRNA with length of 40 nucleotides has been prepared that is necessary and sufficient for the specific interaction with the MjaL4 protein.

High-resolution crystal structure of the isolated ribosomal L1 stalk.

The crystal structure of the isolated full-length ribosomal L1 stalk, consisting of Thermus thermophilus ribosomal protein L1 in complex with a specific 80-nucleotide fragment of 23S rRNA, has been solved for the first time at high resolution. The structure revealed details of protein-RNA interactions in the L1 stalk. Analysis of the crystal packing enabled the identification of sticky sites on the protein and the 23S rRNA which may be important for ribosome assembly and function. The structure was used to model different conformational states of the ribosome. This approach provides an insight into the roles of domain II of L1 and helix 78 of rRNA in ribosome function.

Structural analysis of interdomain mobility in ribosomal L1 proteins.

Ribosomal protein L1 consists of two domains connected by two oppositely directed fragments of the polypeptide chain in a hinge-resembling fashion. The domain arrangement determines the overall shape of the protein, corresponding to an open or a closed conformation. Ribosomal L1 proteins from archaea demonstrate the open conformation in both isolated and RNA-bound forms. RNA-free ribosomal L1 proteins from bacteria display the closed conformation, whereas in complex with RNA these proteins exist in an open conformation similar to their archaeal counterparts. Analysis of all available L1 amino-acid sequences shows that in comparison to the archaeal proteins, the bacterial proteins possess an extra residue in one of the two interdomain fragments which could be responsible for their closed conformation. To verify this suggestion, a Thermus thermophilus L1 mutant lacking one residue in the fragment corresponding to the hinge was obtained and its crystal structure was solved. It was found that this mutation transformed the closed conformation of the bacterial L1 protein into an open conformation similar to that of the archaeal L1 proteins.

[Crystal structures of mutant ribosomal proteins L1].

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.

Structure of the ribosomal protein L1-mRNA complex at 2.1 A resolution: common features of crystal packing of L1-RNA complexes.

The crystal structure of a hybrid complex between the bacterial ribosomal protein L1 from Thermus thermophilus and a Methanococcus vannielii mRNA fragment containing an L1-binding site was determined at 2.1 A resolution. It was found that all polar atoms involved in conserved protein-RNA hydrogen bonds have high values of density in the electron-density map and that their hydrogen-bonding capacity is fully realised through interactions with protein atoms, water molecules and K(+) ions. Intermolecular contacts were thoroughly analyzed in the present crystals and in crystals of previously determined L1-RNA complexes. It was shown that extension of the RNA helices providing canonical helix stacking between open-open or open-closed ends of RNA fragments is a common feature of these and all known crystals of complexes between ribosomal proteins and RNAs. In addition, the overwhelming majority of complexes between ribosomal proteins and RNA molecules display crystal contacts formed by the central parts of the RNA fragments. These contacts are often very extensive and strong and it is proposed that they are formed in the saturated solution prior to crystal formation.

[Interactions of ribosomal protein L1 with ribosomal and messenger RNAs].

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.

Isolation, crystallization, and investigation of ribosomal protein S8 complexed with specific fragments of rRNA of bacterial or archaeal origin.

The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restricted by nucleotides 588-602 and 636-651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA fragments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and synthesized in preparative amounts in vitro using T7 RNA-polymerase. Stable S8-RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bacterial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37-nucleotide rRNA fragment from the same organism suitable for X-ray analysis were obtained.

Detailed analysis of RNA-protein interactions within the ribosomal protein S8-rRNA complex from the archaeon Methanococcus jannaschii.

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.

The crystal structure of UUCG tetraloop.

All large structured RNAs contain hairpin motifs made of a stem closed by several looped nucleotides. The most frequent loop motif is the UUCG one. This motif belongs to the tetraloop family and has the peculiarity of being highly thermodynamically stable. Here, we report the first crystal structure of two UUCG tetraloops embedded in a larger RNA-protein complex solved at 2.8 A resolution. The two loops present in the asymmetric unit are in a different crystal packing environment but, nevertheless, have an identical conformation. The observed structure is globally close to that obtained in solution by nuclear magnetic resonance. However, subtle differences point to a more detailed picture of the role played by 2'-hydroxyl groups in stabilising this tetraloop.

Crystal structure of the S15-rRNA complex.

In bacterial ribosomes, the small (30S) ribosomal subunit is composed of 16S rRNA and 21 distinct proteins. Ribosomal protein S15 is of particular interest because it binds primarily to 16S rRNA and is required for assembly of the small subunit and for intersubunit association, thus representing a key element in the assembly of a whole ribosome. Here we report the 2.8 ¿ resolution crystal structure of the highly conserved S15-rRNA complex. Protein S15 interacts in the minor groove with a G-U/G-C motif and a three-way junction. The latter is constrained by a conserved base triple and stacking interactions, and locked into place by magnesium ions and protein side chains, mainly through interactions with the unique three-dimensional geometry of the backbone. The present structure gives insights into the dual role of S15 in ribosome assembly and translational regulation.

Structure of ribosomal protein L30 from Thermus thermophilus at 1.9 A resolution: conformational flexibility of the molecule.

The crystal structure of ribosomal protein L30 from the extreme thermophilic bacterium Thermus thermophilus has been determined at 1. 9 A resolution. The crystals are trigonal and belong to space group P3(2)21, with unit-cell parameters a = b = 63.5, c = 77.8 A, alpha = beta = 90, gamma = 120 degrees and two molecules per asymmetric unit. The structure was solved by the molecular-replacement method with AMoRe and refined with X-PLOR to an R value of 20.3% and an R(free) of 25.3% in the resolution range 8-1.9 A. Detailed analyses of the structures of the two molecules in the asymmetric unit and comparison of T. thermophilus L30 structure with the structure of homologous L30 from Bacillus stearothermophilus reveal two flexible regions at opposite ends of the rather elongated molecule. Such flexibility could be important for the protein fitting in the ribosome. A comparison with B. stearothermophilus L30 shows a higher number of salt bridges and unbound positively charged residues and an increased accessible hydrophobic area on the surface of T. thermophilus L30. This could contribute to the stability of both the extreme thermophile protein and the ribosome as a whole.