RESUMO
The gatC, gatA and gatB genes encoding the three subunits of glutamyl-tRNA(Gln) amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon-like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu-tRNA(Gln) to Gln-tRNA(Gln) and Asp-tRNA(Asn) to Asn-tRNA(Asn). Biochemical analysis revealed that neither glutaminyl-tRNA synthetase nor asparaginyl-tRNA synthetase is present in A. ferrooxidans, but that glutamyl-tRNA synthetase and aspartyl-tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln-tRNA and Asn-tRNA in A. ferrooxidans.
Assuntos
Asparagina/genética , Aspartato-tRNA Ligase , Gammaproteobacteria/enzimologia , Glutamina/genética , Transferases de Grupos Nitrogenados/metabolismo , Clonagem Molecular , Códon/genética , Ativação Enzimática/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Gammaproteobacteria/genética , Transferases de Grupos Nitrogenados/genética , Biossíntese de Proteínas/fisiologia , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Aminoacil-RNA de Transferência/biossíntese , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Especificidade por Substrato/fisiologiaRESUMO
Bacterial tyrosyl-tRNA synthetases occur in two large subfamilies, TyrRS and TyrRZ, that possess about 25% amino acid identity. Their amino-terminal region, the active site domain, is more conserved (>36% identity). The carboxy-terminal segment of these enzymes includes the tRNA binding domain and contains only few conserved residues. Replacement of three of these residues in Acidithiobacillus ferrooxidans TyrRZ revealed that S356 and K395 play roles in tRNA binding, while H306, a residue at the junction of the catalytic and tRNA binding domains, stabilizes the Tyr-AMP:TyrRZ complex. The replacement data suggest that conserved amino acids in A. ferrooxidans TyrRZ and Bacillus stearothermophilus TyrRS play equivalent roles in enzyme function.
Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Gammaproteobacteria/enzimologia , RNA de Transferência/metabolismo , Tirosina-tRNA Ligase/metabolismo , Tirosina/metabolismo , Monofosfato de Adenosina/análogos & derivados , Proteínas de Bactérias/química , Clonagem Molecular , Sequência Conservada , Dimerização , Escherichia coli/genética , Gammaproteobacteria/genética , Expressão Gênica , Teste de Complementação Genética , Geobacillus stearothermophilus/enzimologia , Mutagênese Sítio-Dirigida , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Tirosina/análogos & derivados , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/genéticaRESUMO
The success of a procedure to reanimate paralyzed eyelids is determined by the functional and cosmetic results. When the cornea is covered during blinking and sleeping, function has been restored, while a pleasing cosmetic result has been achieved if the eyes appear symmetrical when the lids are open. Several procedures have been developed to restore closure of the paralyzed upper eyelid (implantation of gold weights or open wire springs) or to correct lower lid lagophthalmos and ectropion (lower lid tightening with a Bick procedure or insertion of a closed eyelid spring). In some cases, even a combination of the Bick procedure and insertion of a spring may be insufficient to correct lower lid droop; therefore, we developed a technique to place cartilage into the lower eyelid to correct lid droop. The procedure, suggested by one of us (D.B.S.), has been performed on 51 patients to date. This article reviews our experience with these 51 consecutive patients.
Assuntos
Cartilagem da Orelha/transplante , Orelha Externa/transplante , Doenças Palpebrais/cirurgia , Pálpebras/cirurgia , Paralisia Facial/cirurgia , Humanos , Complicações Pós-Operatórias , Reoperação , SuturasRESUMO
We have isolated temperature resistant revertants from temperature sensitive E. coli strains containing either a thermolabile glutaminyl-tRNA synthetase or leucyl-tRNA synthetase. Among the revertants which still contained the thermolabile leucyl-tRNA synthetase we found two classes of regulatory mutants (leuX and leu Y) which have elevated levels of this enzyme. The leuX mutation specifies an operator-promoter region adjacent to the structural gene (leuS) for the enzyme. The leuY gene maps away from the leuS gene and codes for a protein. Using these mutants we demonstrated that the levels of leucyl-tRNA are related to the derepression of the leucine and isoleucine-valine operons. Among the revertants which still contained the thermolabile glutaminyl-tRNA synthetase were characterized three classes of mutants, glnT, glnU, and glnR. The glnT and glnU mutants contain elevated levels of tRNAgln, while the glnR mutant possesses elevated levels of glutaminyl-tRNA synthetase. The level of glutamine synthetase, the enzyme responsible for the formation of glutamine, is also derepressed in the glnT and glnR mutants.