Solution structure of the ribosomal RNA binding protein S15 from Thermus thermophilus.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
M Garber
Publications and source records attributed to M Garber.
Severe sensory deficits but normal CNS development in newborn mice lacking TrkB and TrkC tyrosine protein kinase receptors.
Analysis of mice carrying targeted mutations in genes encoding neurotrophins and their signalling Trk receptors has provided critical information regarding the role that these molecules play in the mammalian nervous system. In this study we generated mice defective in both TrkB and TrkC tyrosine kinase receptors to determine the biological effects of these receptors in the absence of compensatory mechanisms. trkB(-/-);trkC(-/-) double-mutant mice were born at the expected frequency, indicating that TrkB and TrkC signalling are not required for embryonic survival. However, these double-mutant mice had a significantly shorter lifespan and displayed more severe sensory defects than their single-mutant trkB(-/-) and trkC(-/-) littermates. The most dramatic sensory deficit observed in trkB(-/-);trkC(-/-) mutant mice was the absence of vestibular and cochlear ganglia. Interestingly, these mice developed inner ear sensory epithelia in spite of the complete absence of sensory innervation. Analysis of the CNS in trkB(-/-);trkC(-/-) mutant mice revealed a well formed hippocampus, cortex and thalamus. Moreover, the pattern of expression of several neuronal markers appeared normal in these animals. These observations suggest that neurotrophin signalling through TrkB and TrkC receptors is essential for the development of sensory ganglia; however, it does not play a major role in the differentiation and survival of CNS neurons during embryonic development.
Animals↗
5S rRNA binding ribosomal proteins from Thermus thermophilus: identification and some structural properties.
An unusual acidic ribosomal protein from Thermus thermophilus, TL5, that binds to 5S rRNA specifically and strongly, has been investigated. The N-terminal sequence of TL5 does not reveal any homology with known ribosomal proteins. Two large tryptic fragments of TL5 have been isolated and characterized. 5S rRNA protected TL5 and its unstable N-terminal fragment against trypsin action. The 5S rRNA binding ability of TL5 is probably inherent in its N-terminal part. The other 5S rRNA binding ribosomal protein from T. thermophilus, TL4, has been identified as a homolog of the ribosomal protein L5 from Escherichia coli.
Amino Acid Sequence↗
The structure of elongation factor G in complex with GDP: conformational flexibility and nucleotide exchange.
BACKGROUND: Elongation factor G (EF-G) catalyzes the translocation step of translation. During translocation EF-G passes through four main conformational states: the GDP complex, the nucleotide-free state, the GTP complex, and the GTPase conformation. The first two of these conformations have been previously investigated by crystallographic methods. RESULTS: The structure of EF-G-GDP has been refined at 2.4 A resolution. Comparison with the nucleotide-free structure reveals that, upon GDP release, the phosphate-binding loop (P-loop) adopts a closed conformation. This affects the position of helix CG, the switch II loop and domains II, IV and V. Asp83 has a conformation similar to the conformation of the corresponding residue in the EF-Tu/EF-Ts complex. The magnesium ion is absent in EF-G-GDP. CONCLUSIONS: The results illustrate that conformational changes in the P-loop can be transmitted to other parts of the structure. A comparison of the structures of EF-G and EF-Tu suggests that EF-G, like EF-Tu, undergoes a transition with domain rearrangements. The conformation of EF-G-GDP around the nucleotide-binding site may be related to the mechanism of nucleotide exchange.
Binding Sites↗
Crystal structure of the RNA binding ribosomal protein L1 from Thermus thermophilus.
L1 has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding mRNA. The crystal structure of L1 from Thermus thermophilus has been determined at 1.85 angstroms resolution. The protein is composed of two domains with the N- and C-termini in domain I. The eight N-terminal residues are very flexible, as the quality of electron density map shows. Proteolysis experiments have shown that the N-terminal tail is accessible and important for 23S rRNA binding. Most of the conserved amino acids are situated at the interface between the two domains. They probably form the specific RNA binding site of L1. Limited non-covalent contacts between the domains indicate an unstable domain interaction in the present conformation. Domain flexibility and RNA binding by induced fit seems plausible.
Amino Acid Sequence↗
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.
Base Sequence↗
Ribosomal proteins and elongation factors.
Structural work on the translation machinery has recently undergone rapid progress. It is now known that six out of nine ribosomal proteins have an RNA-binding fold, and two domains of elongation factors Tu and G have very similar folds. In addition, the complex of EF-Tu with a GTP analogue and Phe-tRNA(Phe) has a structure that overlaps exceedingly well with that of EF-G-GDP. These findings obviously have functional implications.
Fungal Proteins↗
Low oxygen enhances sickle and normal erythropoiesis and fetal hemoglobin synthesis in vitro.
Erythropoiesis is increased in cultures of human blood progenitors when oxygen tension is reduced from 20% (room air) to 5% (low oxygen, closer to physiological bone marrow levels). The effects of low oxygen on gamma-globin synthesis and colony growth in methyl cellulose cultures of blood mononuclear cells from normal individuals and patients with sickle cell diseases were examined. Low oxygen increased colony numbers by 1.5- to 2-fold and erythropoietin sensitivity by almost 2-fold. The interval required for maximal colony growth in cultures from patients with sickle cell disease (sickle colonies) was reduced from 17 days in 20% oxygen to 13 days in 5% oxygen. Relative synthesis of gamma-globin was examined by labeling with 3H-leucine and electrophoresis on Triton acid urea polyacrylamide gels. The % gamma was 1.7-fold higher in normal and 1.4-fold higher in sickle cultures on day 13 in low oxygen. On day 16 the expected temporal decline was not seen in low oxygen, and the % gamma was 2-fold higher in normal and 1.8-fold higher in the sickle studies. Hemin increased colony growth and gamma-globin synthesis in normal cultures in air, and the effects of hemin and low oxygen were additive. In sickle cultures, hemin and low oxygen had additive effects on colony growth, but only low oxygen increased gamma-globin synthesis. Interleukin-3 increased colony numbers on day 13, primarily by acceleration of peak growth. Interleukin-3 also increased gamma-globin synthesis in low oxygen in normal but not sickle cultures. Thus, low oxygen increases in vitro sensitivity to erythropoietin, colony numbers, and relative gamma-globin synthesis in normal and sickle cultures.
Cell Hypoxia↗
Three-dimensional structure of the ribosomal translocase: elongation factor G from Thermus thermophilus.
The crystal structure of Thermus thermophilus elongation factor G without guanine nucleotide was determined to 2.85 A. This GTPase has five domains with overall dimensions of 50 x 60 x 118 A. The GTP binding domain has a core common to other GTPases with a unique subdomain which probably functions as an intrinsic nucleotide exchange factor. Domains I and II are homologous to elongation factor Tu and their arrangement, both with and without GDP, is more similar to elongation factor Tu in complex with a GTP analogue than with GDP. Domains III and V show structural similarities to ribosomal proteins. Domain IV protrudes from the main body of the protein and has an extraordinary topology with a left-handed cross-over connection between two parallel beta-strands.
Amino Acid Sequence↗
Crystal structure of a prokaryotic aspartyl tRNA-synthetase.
The crystal structure of Thermus thermophilus aspartyl tRNA-synthetase (AspRS) refined at 2.5 A resolution is described. This molecular structure is a textbook illustration of the modular organization of aminoacyl-tRNA synthetases. In addition to the three domains found in yeast AspRS, each monomer exhibits a module specific to prokaryotic enzymes, which corresponds to a helix-turn-helix motif in yeast AspRS, a domain implicated in the stabilization of the complex with tRNA. Its topology matches that of the histidine-containing phosphocarrier HPr which has been linked recently to another group of proteins containing the ferredoxin fold. We propose a more extensive alignment of these folds, which involves a circular permutation of the sequences and changes the point of entry of the whole domain. The C-terminal extension, another prokaryotic characteristic, leads to a significant increase in the network of interaction at the dimer interface. Some potential communication pathways suggest how a transfer of information between the two active sites of the homodimer might occur. Most of the residues involved belong to the class II-specific motifs in correlation with the dimeric state of nearly all class II enzymes. The T. thermophilus enzyme exhibits some features not found in any of the six other known AspRSs from mesophilic organisms.
Amino Acid Sequence↗
The ribosomal protein S8 from Thermus thermophilus VK1. Sequencing of the gene, overexpression of the protein in Escherichia coli and interaction with rRNA.
The gene of the ribosomal protein S8 from Thermus thermophilus VK1 has been isolated from a genomic library by hybridization of an oligonucleotide coding for the N-terminal amino acid sequence of the protein, amplified by PCR and sequenced. Nucleotide sequence reveals an open reading frame coding for a protein of 138 amino acid residues (M(r) 15,839). The codon usage shows that 94% of the codons possess G or C in the third position, and agrees with the preferential usage of codons of high G+C content in the bacteria of the genus Thermus. The amino acid sequence of the protein shows 48% identity with the protein from Escherichia coli. Ribosomal protein S8 from T. thermophilus has been expressed in E. coli under the control of the T7 promoter and purified to homogeneity by heat treatment of the extract followed by cation-exchange chromatography. Conditions were defined in which T. thermophilus protein S8 binds specifically an homologous 16S rRNA fragment containing the putative S8 binding site with an apparent association constant of 5 x 10(7) M-1. The overexpressed protein binds the rRNA with the same affinity as that extracted from T. thermophilus, indicating that the thermophilic protein is correctly folded in E. coli. The specificity of this binding is dependent on the ionic strength. The protein S8 from T. thermophilus recognizes the E. coli rRNA binding sites as efficiently as the S8 protein from E. coli. This result agrees with sequence comparisons of the S8 binding site on the small subunit rRNA from E. coli and from T. thermophilus, showing strong similarities in the regions involved in the interaction. It suggests that the structural features responsible for the recognition are conserved in the mesophilic and thermophilic eubacteria, despite structural peculiarities in the thermophilic partners conferring thermostability.
Amino Acid Sequence↗
Threonyl-tRNA synthetase from Thermus thermophilus: purification and some structural and kinetic properties.
Threonyl-tRNA synthetase (ThrRS) has been isolated from an extreme thermophile Thermus thermophilus strain HB8. The enzyme was purified to electrophoretic homogeneity by combinations of column chromatographies on DEAE-Sepharose, S-Sepharose, ACA-44 Ultrogel and HA-Ultrogel. Seventeen mg of purified enzyme were obtained from 1 kg of biomass. In parallel, purified aspartyl- and phenylalanyl-tRNA synthetases were obtained. The purified ThrRS is composed of two identical subunits with a molecular mass of about 77,000 (virtually the same as E coli ThrRS). The N-terminal sequence has been determined. The homology between the first 45 amino acid residues of ThrRS from T thermophilus and E coli is about 29%. A comparative study of tRNA(Thr) charging by ThrRS from E coli and T thermophilus reveals a similar efficiency of the reaction in both homologous systems. This efficiency remains unchanged for aminoacylation of tRNA(Thr) from T thermophilus by the heterologous ThrRS from E coli, but decreases 700 times for aminoacylation of E coli tRNA(Thr) by ThrRS from T thermophilus.
Amino Acid Sequence↗
The complete amino acid sequence of ribosomal protein S8 from Thermus thermophilus.
Protein S8 from Thermus thermophilus consists of 138 amino acids of M(r) 15,840. Its primary structure was established using peptide sequences from two different digests. Protein S8 from T. thermophilus shares a high percentage of identity with protein S8 from Thermus aquaticus. There are some consensus sequences between proteins S8 from eubacteria, archebacteria, chloroplasts, and cyanelles.
Amino Acid Sequence↗
The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, induces specific transcription by RNA polymerase III in Drosophila Schneider cells.
We have examined the ability of the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), to regulate RNA polymerase III gene expression in Drosophila. Using nuclear run-on assays, we detected a 3-5-fold increase in tRNA synthesis following treatment of Drosophila Schneider S2 cells with TPA, whereas transcription from the actin 5C, fos-, and jun-related antigen promoters was unaffected. This response is rapid and transient, peaking at about a 45-min exposure of the cells to TPA, and dissipating after 60 min. We have reproduced this stimulation in vitro. Extracts prepared from cells treated with TPA show an approximate 10-fold increase in specific transcription using a 5 S RNA and various tRNA gene templates. The nonspecific transcription by RNA polymerase III in these extracts, however, is essentially unchanged. Mixing the extracts derived from uninduced and induced cells suggests that the TPA stimulation observed may be due to the increase of a positive-acting factor. These results are the first to demonstrate that a phorbol ester can induce RNA polymerase III gene expression. The ability to reproduce this activation in vitro will now allow us to assess the role of the transcription components in this regulatory event, and the biochemical consequence of this signaling pathway.
Actins↗
Exploring the experience of autism through firsthand accounts.
Two firsthand accounts from verbal, high-functioning individuals with autism are presented. Participants include a 27-year-old man and a 13-year-old boy and his parents. Data collection included participant observation, formal and informal interviewing, correspondence, and collection of personal documents such as poems, art work, and essays. It was our aim to explore the participants' perceptions, mental processes, and experiences of living with autism. Five salient themes are identified and discussed: sensory processing, memory, stereotypical behaviors, social interaction, and empathy. Multichanneled sensory processing appeared in both participants, as well as a remarkably detailed memory of past events. Both participants were able to bring their stereotypical behaviors under voluntary control as a result of their awareness of nonautistic individuals' reactions to these behaviors.
Adaptation, Psychological↗
Preliminary X-ray investigation of 70 S ribosome crystals from Thermus thermophilus.
Large three-dimensional crystals of 70 S from Thermus thermophilus have been grown from solutions of 2-methyl-2,4-pentanediol at 4 degrees C and examined in an X-ray synchrotron beam. The space group is P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = 510 A and c = 378 A. The diffraction patterns extend to better than 20 A.
Crystallization↗
Carbamate poisoning: the 'other' insecticide.
A 16-month-old child who ingested rat poison, according to her parents, was noted to have signs of cholinergic poisoning. In the emergency department, the child was intubated and given atropine via the endotracheal tube until venous access was established. Phytonadione (vitamin K) and pralidoxime (2-PAM) were also administered. The child recovered after an uneventful hospital course. The toxic agent was determined to be a carbamate insecticide, for which treatment with pralidoxime is considered controversial. Treatment of cholinergic poisoning due to unknown or mixed agents and poisoning caused by known carbamate insecticides are discussed.
Antidotes↗
Hereditary sclerocornea.
Sclerocornea is a primary anomaly in which scleralization of a peripheral part of the cornea, or the entire corneal tissue, occurs. In the peripheral type of sclerocornea, the affected area is vascularized with regular arcades of superficial scleral vessels. In total sclerocornea, the entire cornea is opaque and vascularized. To our knowledge, 97 cases of all types of sclerocornea have been reported in the world literature, either as a primary anomaly or in association with cornea plana. Peripheral sclerocornea in association with cornea plana was found in nine members of one family, in four of five generations studied. To our knowledge, this is the largest pedigree of hereditary peripheral sclerocornea identified. Our pedigree suggests the autosomal-dominant transmission of this entity but doesn't rule out phenocopies or other modes of inheritance in other cases of sclerocornea. Chromosomal analyses of representative family members revealed normal karyotypes.
Adult↗