Premature recruitment of oocyte pool and increased mTOR activity in Fmr1 knockout mice and reversal of phenotype with rapamycin.

While mutations in the fragile X mental retardation-1 (FMR1) gene are associated with varying reproductive outcomes in females, the effects of a complete lack of FMR1 expression are not known. Here, we studied the ovarian and reproductive phenotypes in an Fmr1 knockout (KO) mouse model and the role of mammalian target of rapamycin (mTOR) signaling. Breeding, histologic and mTOR signaling data were obtained at multiple time points in KO and wild type (WT) mice fed a control or rapamycin (mTOR inhibitor) diet. KO mice showed an earlier decline in ovarian reserve than WT mice with an increased proportion of activated follicles. mTOR and phosphorylated S6 kinase (p-S6K) levels, a measure of downstream mTOR signaling, were elevated in the KO ovaries. Rapamycin blocked these effects in KO mice, and increased the primordial follicle pool and age of last litter in WT mice. Our data demonstrates an early decline in reproductive capacity in Fmr1 KO mice and proposes that premature recruitment of the primordial pool via altered mTOR signaling may be the mechanism. Reversal of phenotypes and protein levels in rapamycin-treated KO mice, as well as increased reproductive lifespan of rapamycin-fed WT mice, suggest the mTOR pathway as a potential therapeutic target.

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.

[The Thermus thermophilus 5S rRNA-protein complex: Identifications of specific binding sites for proteins L5 and L18 in 5S rRNA]. / 5S rRNK-belkovyi kompleks Thermus thermophilus. Opredelenie spetsificheskikh uchastkov sviazyvaniia belkov L5 i L18 na 5S rRNK.

Three 5S rRNA-binding ribosomal proteins (L5, L18, TL5) of extremely thermophilic bacterium Thermus thermophilus have earlier been isolated. Structural analysis of their complexes with rRNA requires identification of their binding sites in the 5S rRNA. Previously, a TL5-binding site has been identified, a TL5-RNA complex crystallized, and its structure determined to 2.3 A. The sites for L5 and L18 were characterized, and two corresponding 5S rRNA fragments constructed. Of these, a 34-nt fragment specifically interacted with L5, and a 55-nt fragment interacted with L5, L18, and with both proteins. The 34-nt fragment-L5 complex was crystallized; the crystals are suitable for high-resolution X-ray analysis.

Role of conserved nucleotides in building the 16 S rRNA binding site for ribosomal protein S15.

Ribosomal protein S15 recognizes a highly conserved target on 16 S rRNA, which consists of two distinct binding regions. Here, we used extensive site-directed mutagenesis on a Escherichia coli 16 S rRNA fragment containing the S15 binding site, to investigate the role of conserved nucleotides in protein recognition and to evaluate the relative contribution of the two sites. The effect of mutations on S15 recognition was studied by measuring the relative binding affinity, RNA probing and footprinting. The crystallographic structure of the Thermus thermophilus complex allowed molecular modelling of the E. coli complex and facilitated interpretation of biochemical data. Binding is essentially driven by site 1, which includes a three-way junction constrained by a conserved base triple and cross-strand stacking. Recognition is based mainly on shape complementarity, and the role of conserved nucleotides is to maintain a unique backbone geometry. The wild-type base triple is absolutely required for protein interaction, while changes in the conserved surrounding nucleotides are partially tolerated. Site 2, which provides functional groups in a conserved G-U/G-C motif, contributes only modestly to the stability of the interaction. Binding to this motif is dependent on binding at site 1 and is allowed only if the two sites are in the correct relative orientation. Non-conserved bulged nucleotides as well as a conserved purine interior loop, although not directly involved in recognition, are used to provide an appropriate flexibility between the two sites. In addition, correct binding at the two sites triggers conformational adjustments in the purine interior loop and in a distal region, which are known to be involved for subsequent binding of proteins S6 and S18. Thus, the role of site 1 is to anchor S15 to the rRNA, while binding at site 2 is aimed to induce a cascade of events required for subunit assembly.

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.

N-terminal domain, residues 1-91, of ribosomal protein TL5 from Thermus thermophilus binds specifically and strongly to the region of 5S rRNA containing loop E.

In this work we show for the first time that the overproduced N-terminal fragment (residues 1-91) of ribosomal protein TL5 binds specifically to 5S rRNA and that the region of this fragment containing residues 80-91 is a necessity for its RNA-binding activity. The fragment of Escherichia coli 5S rRNA protected by TL5 against RNase A hydrolysis was isolated and sequenced. This 39 nucleotides fragment contains loop E and helices IV and V of 5S rRNA. The isolated RNA fragment forms stable complexes with TL5 and its N-terminal domain. Crystals of TL5 in complex with the RNA fragment diffracting to 2.75 A resolution were obtained.

Ribosomal protein S15 from Thermus thermophilus--cloning, sequencing, overexpression of the gene and RNA-binding properties of the protein.

A 6-kb DNA fragment from an extreme thermophile, Thermus thermophilus, carrying the genes for cytochrome oxidase ba3 subunit I (cbaA) and the ribosomal protein S15 (rpsO) was cloned into Escherichia coli. The gene rpsO was sequenced. The deduced amino acid sequence exhibits 59% identity to the corresponding protein from E. coli. Expression of rpsO in E. coli requires the use of a fully repressed inducible promoter because S15 from T. thermophilus is toxic for E. coli cells. When purified without denaturation from either overproducing E. coli strain or from T. thermophilus ribosomes, the S15 protein is stable and binds a cloned T. thermophilus 16S rRNA fragment (nucleotides 559-753), with low identical dissociation constants (2.5 nM), thus demonstrating that the thermophilic protein folds correctly in a mesophilic bacterium. The rRNA fragment bound corresponds in position and structure to the 16S rRNA fragment of E. coli. A similar high affinity was also found for the binding of S15 from T. thermophilus or E. coli to the corresponding E. coli 16S rRNA fragment, whereas a slightly lower affinity was observed in binding experiments between E. coli S15 and T. thermophilus 16S rRNA fragment. These results suggest that S15 from T. thermophilus recognizes similar determinants in both rRNA fragments. Competition experiments support this conclusion.

The 16S rRNA binding site of Thermus thermophilus ribosomal protein S15: comparison with Escherichia coli S15, minimum site and structure.

Binding of Escherichia coli and Thermus thermophilus ribosomal proteins S15 to a 16S ribosomal RNA fragment from T. thermophilus (nt 559-753) has been investigated in detail by extensive deletion analysis, filter-binding assays, gel mobility shift, structure probing, footprinting with chemical, enzymatic, and hydroxyl radical probes. Both S15 proteins recognize two distinct sites. The first one maps in the bottom of helix 638-655/717-734 (H22) and in the three-way junction between helix 560-570/737-747 (H20), helix 571-600/606-634 (H21), and H22. The second is located in a conserved purine-rich region in the center of H22. The first site provides a higher contribution to the free energy of binding than the second one, and both are required for efficient binding. A short RNA fragment of 56 nt containing these elements binds S15 with high affinity. The structure of the rRNA is constrained by the three-way junction and requires both magnesium and S15 to be stabilized. A 3D model, derived by computer modeling with the use of experimental data, suggests that the bound form adopts a Y-shaped conformation, with a quasi-coaxial stacking of H22 on H20, and H21 forming an acute angle with H22. In this model, S15 binds to the shallow groove of the RNA on the exterior side of the Y-shaped structure, making contact with the two sites, which are separated by one helix turn.

A radioimmunoassay-based method for measuring the true affinity of a monoclonal antibody with trace amounts of radioactive antigen: illustration with the products of a cell-free protein synthesis system.

This communication describes a novel highly sensitive method for measuring the affinity of a monoclonal antibody for its antigen. It is based on a radioimmunoassay in which the antigen is labeled with radioactivity. It is therefore particularly well adapted to the study of trace amounts of radiolabeled polypeptide chains produced either in vivo, or in vitro by a cell free protein synthesis system or by chemical radiolabeling. It offers several advantages over previously described methods. Though making use of insolubilized antibody, it does measure the true affinity constant of the monoclonal antibody in solution for the antigen. It can be used even when the antigen is present at concentrations far below the dissociation constant of the antibody/antigen complex. It does not require the antigen or the antibody to be purified. In most cases, it requires no sophisticated equipment. This method could be easily adapted to the determination of the equilibrium constant of any type of protein/ligand system.