RÉSUMÉ
The heterotrimeric (αßγ) translation initiation factor 2 of archaea and eukaryotes (a/eIF2) supplies the P-site of the ribosome with the initiation tRNA. Its two subunits (ß 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 aIF2ß 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 ß- and γ-subunits of the aIF2 are different.
Sujet(s)
Protéines d'archée/composition chimique , Cystéine/composition chimique , Facteurs initiation chaîne peptidique/composition chimique , Facteur d'initiation procaryote 2/composition chimique , Sites de fixation , Cristallographie aux rayons X , Humains , Ions , Conformation moléculaire , Conformation des protéines , Multimérisation de protéines , Structure secondaire des protéines , Sous-unités de protéines/composition chimique , Sulfolobus solfataricus/composition chimique , Zinc , Doigts de zincRÉSUMÉ
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.
Sujet(s)
Protéines du cycle cellulaire/composition chimique , Facteurs d'initiation eucaryotes/composition chimique , Protéines oncogènes/composition chimique , Ribosomes/composition chimique , Cristallographie aux rayons X , Humains , Structure quaternaire des protéinesRÉSUMÉ
mRNAs lacking 5' untranslated regions (leaderless mRNAs) are molecular relics of an ancient translation initiation pathway. Nevertheless, they still represent a significant portion of transcriptome in some taxons, including a number of eukaryotic species. In bacteria and archaea, the leaderless mRNAs can bind non-dissociated 70 S ribosomes and initiate translation without protein initiation factors involved. Here we use the Fleeting mRNA Transfection technique (FLERT) to show that translation of a leaderless reporter mRNA is resistant to conditions when eIF2 and eIF4F, two key eukaryotic translation initiation factors, are inactivated in mammalian cells. We report an unconventional translation initiation pathway utilized by the leaderless mRNA in vitro, in addition to the previously described 80S-, eIF2-, or eIF2D-mediated modes. This mechanism is a bacterial-like eIF5B/IF2-assisted initiation that has only been reported for hepatitis C virus-like internal ribosome entry sites (IRESs). Therefore, the leaderless mRNA is able to take any of four different translation initiation pathways in eukaryotes.
Sujet(s)
Cellules eucaryotes/physiologie , Initiation de la traduction/physiologie , ARN messager/métabolisme , Système acellulaire , Facteur-2 d'initiation eucaryote/génétique , Facteur-2 d'initiation eucaryote/métabolisme , Facteurs d'initiation eucaryotes/génétique , Facteurs d'initiation eucaryotes/métabolisme , Cellules HEK293 , Hépatite C/génétique , Humains , Sites internes d'entrée des ribosomes , Complexes multiprotéiques , Biosynthèse des protéines , ARN messager/génétique , Saccharomyces cerevisiae/génétique , Transfection/méthodesRÉSUMÉ
L1 is a conserved protein of the large ribosomal subunit. This protein binds strongly to the specific region of the high molecular weight rRNA of the large ribosomal subunit, thus forming a conserved flexible structural element--the L1 stalk. L1 protein also regulates translation of the operon that comprises its own gene. Crystallographic data suggest that L1 interacts with RNA mainly by means of its domain I. We show here for the first time that the isolated domain I of the bacterial protein L1 of Thermus thermophilus and Escherichia coli is able to incorporate in vivo into the E. coli ribosome. Furthermore, domain I of T. thermophilus L1 can regulate expression of the L1 gene operon of Archaea in the coupled transcription-translation system in vitro, as well as the intact protein. We have identified the structural elements of domain I of the L1 protein that may be responsible for its regulatory properties.
Sujet(s)
Protéines bactériennes/composition chimique , Opéron/génétique , ARN bactérien/composition chimique , Protéines ribosomiques/composition chimique , Ribosomes/composition chimique , Protéines d'archée/composition chimique , Protéines d'archée/génétique , Protéines bactériennes/génétique , Séquence nucléotidique , Escherichia coli/composition chimique , Escherichia coli/génétique , Données de séquences moléculaires , Plasmides , Structure tertiaire des protéines , ARN bactérien/génétique , ARN ribosomique 23S/composition chimique , ARN ribosomique 23S/génétique , Protéines ribosomiques/génétique , Résonance plasmonique de surface , Thermus thermophilus/composition chimique , Thermus thermophilus/génétiqueRÉSUMÉ
In the present work, ribosomes assembled in bacterial cells in the absence of essential ribosomal protein L5 were obtained. After arresting L5 synthesis, Escherichia coli cells divide a limited number of times. During this time, accumulation of defective large ribosomal subunits occurs. These 45S particles lack most of the central protuberance (CP) components (5S rRNA and proteins L5, L16, L18, L25, L27, L31, L33 and L35) and are not able to associate with the small ribosomal subunit. At the same time, 5S rRNA is found in the cytoplasm in complex with ribosomal proteins L18 and L25 at quantities equal to the amount of ribosomes. Thus, it is the first demonstration that protein L5 plays a key role in formation of the CP during assembly of the large ribosomal subunit in the bacterial cell. A possible model for the CP assembly in vivo is discussed in view of the data obtained.
Sujet(s)
Protéines Escherichia coli/physiologie , Protéines ribosomiques/physiologie , Grande sous-unité du ribosome des bactéries/composition chimique , Escherichia coli/génétique , Escherichia coli/croissance et développement , Protéines Escherichia coli/génétique , Délétion de gène , Modèles moléculaires , ARN ribosomique 5S/composition chimique , Protéines ribosomiques/composition chimique , Protéines ribosomiques/génétique , Grande sous-unité du ribosome des bactéries/métabolisme , Ribosomes/métabolismeRÉSUMÉ
It has become clear that water should not be treated as an inert environment, but rather as an integral and active component of molecules. Here, we consider molecules and their hydration shells together as single entities. We show that: (1) the rate of association of molecules should be determined by the energetic barriers arising from interactions between their hydration shells; (2) replacing non-polar atoms of molecular surfaces with polar atoms increases these barriers; (3) reduction of the hydration shells during molecular association is the driving force for association not only of non-polar, but of polar molecules as well; (4) in most cases the dehydration of polar atoms during molecular association thermodynamically counteracts association; (5) on balance the thermodynamic stability of associated complexes is basically determined by the action of these two opposing factors: reduction of the hydration shells and dehydration of polar atoms; (6) molecular crowding reduces the energetic barriers counteracting association and changes the thermodynamic stability of associated complexes. These results lead to a mechanism for biomolecular recognition in the context of which the formation of unique structures is provided by rapidly forming kinetic traps with a biologically necessary lifetime but with a marginal thermodynamic stability. The mechanism gives definitive answers to questions concerning the heart of specific interactions between biomolecules, their folding and intracellular organization. Predictions are given that can be subjected to direct experimental tests.
Sujet(s)
Modèles moléculaires , Eau/composition chimique , Liaison hydrogène , Pliage des protéines , Propriétés de surface , ThermodynamiqueRÉSUMÉ
Heterotrimeric translation initiation factor (IF) a/eIF2 (archaeal/eukaryotic IF 2) is present in both Eukarya and Archaea. Despite strong structural similarity between a/eIF2 orthologs from the two domains of life, their functional relationship is obscure. Here, we show that aIF2 from Sulfolobus solfataricus can substitute for its mammalian counterpart in the reconstitution of eukaryotic 48S initiation complexes from purified components. aIF2 is able to correctly place the initiator Met-tRNA(i) into the P-site of the 40S ribosomal subunit and accompany the entire set of eukaryotic translation IFs in the process of cap-dependent scanning and AUG codon selection. However, it seems to be unable to participate in the following step of ribosomal subunit joining. In accordance with this, aIF2 inhibits rather than stimulates protein synthesis in mammalian cell-free system. The ability of recombinant aIF2 protein to direct ribosomal scanning suggests that some archaeal mRNAs may utilize this mechanism during translation initiation.
Sujet(s)
Facteurs initiation chaîne peptidique/métabolisme , Biosynthèse des protéines , Ribosomes/métabolisme , Sulfolobus solfataricus/métabolisme , Cellules HeLa , Humains , ARN de transfert de la méthionine/métabolismeRÉSUMÉ
A specific complex of 5 S rRNA and several ribosomal proteins is an integral part of ribosomes in all living organisms. Here we studied the importance of Escherichia coli genes rplE, rplR and rplY, encoding 5 S rRNA-binding ribosomal proteins L5, L18 and L25, respectively, for cell growth, viability and translation. Using recombineering to create gene replacements in the E. coli chromosome, it was shown that rplE and rplR are essential for cell viability, whereas cells deleted for rplY are viable, but grow noticeably slower than the parental strain. The slow growth of these L25-defective cells can be stimulated by a plasmid expressing the rplY gene and also by a plasmid bearing the gene for homologous to L25 general stress protein CTC from Bacillus subtilis. The rplY mutant ribosomes are physically normal and contain all ribosomal proteins except L25. The ribosomes from L25-defective and parental cells translate in vitro at the same rate either poly(U) or natural mRNA. The difference observed was that the mutant ribosomes synthesized less natural polypeptide, compared to wild-type ribosomes both in vivo and in vitro. We speculate that the defect is at the ribosome recycling step.
Sujet(s)
Escherichia coli/métabolisme , Biosynthèse des protéines , ARN bactérien/métabolisme , ARN ribosomique 5S/métabolisme , Protéines ribosomiques/métabolisme , Ribosomes/métabolisme , Survie cellulaire , Clonage moléculaire , Escherichia coli/génétique , Escherichia coli/physiologie , Protéines Escherichia coli/génétique , Mutation , ARN bactérien/génétique , Protéines de répression/génétique , Protéines ribosomiques/génétique , Ribosomes/génétique , Facteurs de transcription/génétiqueRÉSUMÉ
Assembly of 30S ribosomal subunits from Escherichia coli has been dissected in detail using an in vitro system. Such studies have allowed characterization of the role for ribosomal protein S15 in the hierarchical assembly of 30S subunits; S15 is a primary binding protein that orchestrates the assembly of ribosomal proteins S6, S11, S18, and S21 with the central domain of 16S ribosomal RNA to form the platform of the 30S subunit. In vitro S15 is the sole primary binding protein in this cascade, performing a critical role during assembly of these four proteins. To investigate the role of S15 in vivo, the essential nature of rpsO, the gene encoding S15, was examined. Surprisingly, E. coli with an in-frame deletion of rpsO are viable, although at 37 degrees C this DeltarpsO strain has an exaggerated doubling time compared to its parental strain. In the absence of S15, the remaining four platform proteins are assembled into ribosomes in vivo, and the overall architecture of the 30S subunits formed in the DeltarpsO strain at 37 degrees C is not altered. Nonetheless, 30S subunits lacking S15 appear to be somewhat defective in subunit association in vivo and in vitro. In addition, this strain is cold sensitive, displaying a marked ribosome biogenesis defect at low temperature, suggesting that under nonideal conditions S15 is critical for assembly. The viability of this strain indicates that in vivo functional populations of 70S ribosomes must form in the absence of S15 and that 30S subunit assembly has a plasicity that has not previously been revealed or characterized.
Sujet(s)
Protéines ribosomiques/composition chimique , Protéines ribosomiques/métabolisme , Séquence nucléotidique , Amorces ADN , Électrophorèse bidimensionnelle sur gel , Escherichia coli/génétique , Modèles moléculaires , Données de séquences moléculaires , Conformation des protéines , Sous-unités de protéines , ARN bactérien/composition chimique , ARN bactérien/métabolisme , Protéines ribosomiques/isolement et purificationRÉSUMÉ
As a preface to an analysis of the ribosomal elongation cycle, we examine the energetics of macromolecular structural transformations. We show that the kinetic barriers and changes of the energetic levels during these transformations are essentially determined by disruption of hydrogen and cation-ligand bonds, and by uncompensated losses of these bonds (ULBs). The disruption of a hydrogen or cation-ligand bond increases the heights of kinetic barriers by the energy of these bonds. The association and dissociation of macromolecules, and conformational transitions within macromolecules, can change the numbers of ULBs but cannot completely eliminate them. Two important general conclusions are drawn from this analysis. First, occupation of enzyme active centers by substrates should be accompanied by a reduction in the number of ULBs. This reduction decreases the activation barriers in enzyme reactions, and is a major contributor to catalysis. Second, the enzymic reactions of the ribosomal cycle (structural changes caused by transpeptidation and by GTP hydrolyses in EF-Tu and EF-G) disrupt kinetic traps that prevent tRNAs from dissociating into solution during their motion within the ribosome and are necessary for progression of the cycle. These results are general purpose structural-functional blocks for building a molecular model of the ribosomal elongation cycle. Here, we demonstrate the utility of these blocks for analysis of acceptance of cognate tRNAs into the ribosomal elongation cycle.
Sujet(s)
Ribosomes/métabolisme , Cations , Codon , Liaison hydrogène , Cinétique , ARN de transfert/composition chimique , Ribosomes/composition chimique , StéréoisomérieRÉSUMÉ
Analysis of the structures of two complexes of 5 S rRNA with homologous ribosomal proteins, Escherichia coli L25 and Thermus thermophilus TL5, revealed that amino acid residues interacting with RNA can be divided into two different groups. The first group consists of non-conserved residues, which form intermolecular hydrogen bonds accessible to solvent. The second group, comprised of strongly conserved residues, form intermolecular hydrogen bonds that are shielded from solvent. Site-directed mutagenesis was used to introduce mutations into the RNA-binding site of protein TL5. We found that replacement of residues of the first group does not influence the stability of the TL5.5 S rRNA complex, whereas replacement of residues of the second group leads to destabilization or disruption of the complex. Stereochemical analysis shows that the replacements of residues of the second group always create complexes with uncompensated losses of intermolecular hydrogen bonds. We suggest that these shielded intermolecular hydrogen bonds are responsible for the recognition between the protein and RNA.
Sujet(s)
Protéines bactériennes/métabolisme , ARN ribosomique 5S/métabolisme , Protéines de liaison à l'ARN/métabolisme , Protéines ribosomiques/métabolisme , Protéines bactériennes/génétique , Escherichia coli/métabolisme , Liaison hydrogène , Conformation d'acide nucléique , Structure tertiaire des protéines , Protéines de liaison à l'ARN/génétique , Protéines ribosomiques/génétique , Thermus thermophilus/génétique , Thermus thermophilus/métabolismeRÉSUMÉ
Bacterial ribosomes stalled on defective mRNAs are rescued by tmRNA that functions as both tRNA and mRNA. The first ribosomal elongation cycle on tmRNA where tmRNA functions as tRNA is highly unusual: occupation of the ribosomal A site by tmRNA occurs without codon:anticodon pairing. Our analysis shows that in this case the role of a codon:anticodon duplex should be accomplished by a single unpaired triplet. In order that tmRNA could participate in the ribosomal elongation cycle, a triplet preceding the mRNA portion of tmRNA (the -1triplet) should be in the A-form and this form should be recognized by the ribosomal decoding center. A rule is derived that determines what triplets cannot be used as the -1triplet. The rule was tested with the -1triplets of all known 414 tmRNA species. All 23 observed -1triplets follow the formulated rule. The rule is also supported by the available data on base substitutions within the -1triplet.
Sujet(s)
Codon , ARN bactérien/physiologie , Ribosomes/génétique , Sites de fixation , Liaison hydrogène , Modèles moléculaires , Modèles théoriques , Électricité statiqueRÉSUMÉ
Uridine phosphorylase (UPh) catalyzes the phosphorolytic cleavage of the C-N glycosidic bond of uridine to ribose 1-phosphate and uracil in the pyrimidine-salvage pathway. The crystal structure of the Salmonella typhimurium uridine phosphorylase (StUPh) has been determined at 2.5 A resolution and refined to an R factor of 22.1% and an Rfree of 27.9%. The hexameric StUPh displays 32 point-group symmetry and utilizes both twofold and threefold non-crystallographic axes. A phosphate is bound at the active site and forms hydrogen bonds to Arg91, Arg30, Thr94 and Gly26 of one monomer and Arg48 of an adjacent monomer. The hexameric StUPh model reveals a close structural relationship to Escherichia coli uridine phosphorylase (EcUPh).
Sujet(s)
Salmonella typhimurium/enzymologie , Uridine phosphorylase/composition chimique , Sites de fixation , Cristallographie aux rayons X , Escherichia coli/enzymologie , Liaison hydrogène , Structure moléculaire , Conformation des protéinesRÉSUMÉ
Hfq protein from Escherichia coli (EcoHfq) has been overproduced in E. coli, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion technique. Crystallization conditions for EcoHfq were found which yielded X-ray quality crystals. Crystals of EcoHfq and of Cd-, Hg- and Se-containing derivatives grew in two months, with unit-cell parameters a = b = 127.41, c = 170.36 A. The crystals belong to space group I4 and diffract to 2.1 A resolution. Two hexamers are predicted per asymmetric unit.
Sujet(s)
Protéines Escherichia coli/composition chimique , Escherichia coli/génétique , Escherichia coli/métabolisme , Régulation de l'expression des gènes bactériens/génétique , Protéine IHF-1/composition chimique , Clonage moléculaire , Cristallisation , Cristallographie aux rayons X , Protéines Escherichia coli/génétique , Protéine IHF-1/génétique , ARN bactérien/biosynthèse , ARN bactérien/génétiqueRÉSUMÉ
The ribosomal protein L23 is a component of the large ribosomal subunit in which it is located close to the peptide exit tunnel. In this position L23 plays a central role both for protein secretion and folding. We have determined the solution structure of L23 from Thermus thermophilus. Uncomplexed L23 consists of a well-ordered part, with four anti-parallel beta-strands and three alpha-helices connected as beta-alpha-beta-alpha-beta-beta-alpha, and a large and flexible loop inserted between the third and fourth beta-strand. The observed topology is distantly related to previously known structures, primarily within the area of RNA biochemistry. A comparison with RNA-complexed crystal structures of L23 from T. thermophilus, Deinococcus radiodurans and Haloarcula marismourtui, shows that the conformation of the well-ordered part is very similar in the uncomplexed and complexed states. However, the flexible loop found in the uncomplexed solution structure forms a rigid extended structure in the complexed crystal structures as it interacts with rRNA and becomes part of the exit tunnel wall. Structural characteristics of importance for the interaction with rRNA and with the ribosomal protein L29, as well as the functional role of L23, are discussed.
Sujet(s)
Résonance magnétique nucléaire biomoléculaire/méthodes , Protéines ribosomiques/composition chimique , Thermus thermophilus/composition chimique , Modèles moléculaires , Liaison aux protéines , Conformation des protéines , Pliage des protéines , ARN ribosomique/métabolisme , Protéines ribosomiques/métabolisme , Alignement de séquences , Similitude de séquences d'acides aminésRÉSUMÉ
We have determined the solution structure of ribosomal protein L18 from Thermus thermophilus. L18 is a 12.5 kDa protein of the large subunit of the ribosome and binds to both 5 S and 23 S rRNA. In the uncomplexed state L18 folds to a mixed alpha/beta globular structure with a long disordered N-terminal region. We compared our high-resolution structure with RNA-complexed L18 from Haloarcula marismortui and T. thermophilus to examine RNA-induced as well as species-dependent structural differences. We also identified T. thermophilus S11 as a structural homologue and found that the structures of the RNA-recognition sites are conserved. Important features, for instance a bulge in the RNA-contacting beta-sheet, are conserved in both proteins. We suggest that the L18 fold recognizes a specific RNA motif and that the resulting RNA-protein-recognition module is tolerant to variations in sequence.