RESUMEN
Maintenance of amino acid specificity by aminoacyl-tRNA synthetases, particularly prolyl-tRNA synthetase, requires for not only specific recognition of homologic amino acid, but also missynthesized products hydrolysis, known as editing. The speeding-up of the enzymatic hydrolysis of missynthesized alanyl adenylate by bacteria Enterococcus faecalis prolyl-tRNA synthetase in the presence of tRNAPro, and also importance for this function of 2'- and 3'-hydroxyle groups of tRNA 3'-terminal adenosine ribose is shown in the work. Furthermore, results are shown, that support the absence of editing (INS) domain role in alanyl adenylate hydrolysis. Possible significance of tRNA-dependent alanyl adenylate hydrolysis by prolyl-tRNA synthetase for prolyl-tRNAPro synthesis specificity maintenance is discussed.
Asunto(s)
Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Aminoacil-ARNt Sintetasas/genética , Enterococcus faecalis/enzimología , Edición de ARN , ARN de Transferencia/genética , Aminoacilación de ARN de Transferencia , Adenosina Trifosfato/metabolismo , Enterococcus faecalis/genética , Hidrólisis , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Rhodopseudomonas/enzimología , Rhodopseudomonas/genéticaRESUMEN
The reactivity of phosphates in the Thermus thermophilus tRNA(Ser) (GCU) and tRNA(Leu) (CAG) was studied using the ethylnitrosourea modification. It was shown that phosphates of nucleotides 58-60 (T loop), 20-22 (D loop), and 48 (at the junction of the variable and T stems) were poorly modified in both tRNAs. The most pronounced differences in the reactivity were observed for phosphates at the junctions of the variable stem with T-stem (47q, 49) and anticodon stem (45). This indicates differences in orientations of the long variable arm relative to the backbone in the tRNAs studied.
Asunto(s)
Ácidos Fosfóricos/química , ARN de Transferencia de Leucina/química , ARN de Transferencia de Serina/química , Thermus thermophilus/genética , Anticodón , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN de Transferencia de Leucina/genética , ARN de Transferencia de Serina/genéticaRESUMEN
A nucleotide sequence of tRNA(Tyr) from the extreme thermophile Thermus thermophilus HB-27 living at 75 degrees C was determined. It is 86 nt long and shares a 52% homology with tRNA(Tyr) from Escherichia coli. A comparative analysis of the interaction sites of tRNA(Tyr) from T. thermophilus and E. coli with the cognate aminoacyl-tRNA synthetases was accomplished by the chemical modification and nuclease hydrolysis approaches. The tRNA(Tyr) was shown to interact with the cognate enzyme in the anticodon stem (on the 5'-side), in the anticodon, in the variable stem and loop (on the 5'-side), and in the acceptor stem (on the 3'-side). These regions are located in the variable stem of the L-form. It was demonstrated that, upon forming the complex E. coli tRNA(Tyr)-cognate synthetase, endonuclease V1 induces additional cleavages of phosphodiester bonds on the 3'-side of the anticodon stem and on the 5'-side of the T-stem. This implies that tRNA may change its conformation when it interacts with the enzyme.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Escherichia coli/química , ARN Bacteriano/química , ARN de Transferencia de Tirosina/química , Ribonucleasas/química , Thermus thermophilus/química , Autorradiografía , Secuencia de Bases , Escherichia coli/genética , Hidrólisis , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Thermus thermophilus/genéticaRESUMEN
The nucleotide sequence of the tRNALeuIAG from a lactating cow mammary gland was determined by ultramicrospectrophotometrical method and rapid gel sequencing procedure. The chain length of this tRNA is 85 nucleotides, 15 of them including 6 psi, are modified nucleotides. The primary structure of tRNALeuIAG is absolutely identical to major species of leucyl tRNAIAG from bovine liver and differs in 21 positions from cow mammary gland tRNALeuCAG.
Asunto(s)
Lactancia , Glándulas Mamarias Animales/análisis , Aminoacil-ARN de Transferencia/análisis , Animales , Autorradiografía , Secuencia de Bases , Bovinos , Cromatografía en Agarosa , Cromatografía por Intercambio Iónico , Femenino , Hidrólisis , Oligonucleótidos/análisis , EmbarazoRESUMEN
The functional properties of total hemoglobin and some of its major fractions obtained by polyacrylamide gel disk-electrophoresis were studied. It was shown that p50 for intact hemoglobin is 1.18, that for fractions 2, 4-6-1.37-1.65 mm Hg. The affinity of fraction 3 for oxygen is significantly higher. In the presence of CO2 (40 mm Hg) p50 is much higher for fractions 4 and 6 as compared with fractions 3 and 5. Fraction 3 reveals the highest resistance to autooxidation; fractions 2 and 5 possess the highest sensitivity. ATP and pyridoxal-5'-phosphate stimulate the autooxidation and decrease the affinity for hemoglobin oxygen of fractions 3 and 4 in a greater degree than of the other fractions (for fraction 3 more than for fraction 4). A similar dependence is observed in case of fractions 5 and 6. The effective constant (ki) of dissociation of heme- and globin bonds for HbCO for fraction 3 is 2.38.10(-3) s-1. For the other fractions this value is considerably lower. The ki value for all fractions is increased with a transition from the carboxy- to methehemoglobin. The level of individual components of the heterogenous system of hemoglobin in the blood of embryos, adult intact rats and in anemic and hypoxic animals is given. The interrelationship of functional and structural properties of the hemoglobin fractions and their role in oxygen supply of tissues under the above-described conditions are discussed.