RESUMO
Growth kinetics and diffraction properties of monoclinic crystals of eubacterial Thermus thermophilus aspartyl-tRNA synthetase-1 (AspRS-1) prepared in the presence of polyethylene glycol and agarose are studied. Their solubility and two-dimensional phase diagram are compared with those of orthorhombic crystals which grow in the presence of sodium formate or ammonium sulfate. The growth mechanism of the novel crystals was monitored by atomic force microscopy. The gel stabilizes the crystal lattice under the cryogenic conditions used for structure determination at high resolution.
Assuntos
Aspartato-tRNA Ligase/química , Aspartato-tRNA Ligase/metabolismo , Sefarose/metabolismo , Thermus thermophilus/enzimologia , Cristalização , Cristalografia por Raios X/métodos , Estabilidade Enzimática , Géis , Cinética , Microscopia de Força Atômica , Concentração Osmolar , Solubilidade , Temperatura , TermodinâmicaRESUMO
We have solved to 3.3 A resolution the crystal structure of the HIV reverse-transcription primer tRNA(Lys,3). The overall structure is exactly comparable to the well-known L-shape structure first revealed by yeast tRNA(Phe). In particular, it unambiguously shows a canonical anticodon loop. This contradicts previous results in short RNA fragment studies and leads us to conclude that neither frameshifting specificities of tRNA(Lys) nor tRNA(Lys,3) primer selection by HIV are due to a specific three-dimensional anticodon structure. Comparison of our structure with the results of an NMR study on a hairpin representing a nonmodified anticodon stem-loop makes plausible the conclusion that chemical modifications of the wobble base U34 to 5-methoxycarbonyl-methyl-2-thiouridine and of A37 to 2-methylthio-N-6-threonylcarbamoyl-adenosine would be responsible for a canonical 7-nt anticodon-loop structure, whereas the unmodified form would result in a noncanonical UUU short triloop. The hexagonal crystal packing is remarkable and shows tight dimers of tRNAs forming a right-handed double superhelix. Within the dimers, the tRNAs are associated head-to-tail such that the CCA end of one tRNA interacts with the anticodon of the symmetry-related tRNA. This provides us with a partial view of a codon-anticodon interaction and gives insights into the positioning of residue 37, and of its posttranscriptional modifications, relative to the first base of the codon.
Assuntos
Anticódon/química , Galinhas/genética , HIV-1/genética , Conformação de Ácido Nucleico , RNA de Transferência de Lisina/química , RNA/química , Animais , Anticódon/genética , Sequência de Bases , Bovinos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , RNA/genética , RNA de Transferência de Lisina/genética , CoelhosRESUMO
The influence of parathyroid hormone (PTH) as a potential toxin responsible for the anemia in hemodialysed patients is suggested by the fact that 6 patients out of 12 showed improvement of their hematocrit four months after parathyroidectomy. Blood iron levels as well as calcemia, phosphoremia and hematocrit before surgery are not indicative of improvement following the intervention. A direct toxicity of PTH on erythropoiesis seems likely.
