RESUMEN
The ability of the isolated carboxy-terminal domain of the LexA repressor of Escherichia coli to form dimers and tetramers has been investigated by equilibrium ultracentrifugation. This domain, that comprises the amino acids 85-202, is readily purified after self-cleavage of the LexA repressor at alkaline pH. It turns out that the carboxy-terminal domain forms dimers and tetramers essentially as the entire LexA repressor. The corresponding association constants were determined after non-linear least squares fitting of the experimental concentration distribution. A dimer association constant of K2 = 3 X 10(4) M-1 and a tetramer association constant of K4 = 2 X 10(4) M-1 have been determined. Similar measurements on the entire LexA repressor [(1985) Biochemistry 24, 2812-2818] gave values of K2 = 2.1 X 10(4) M-1 and K4 = 7.7 X 10(4) M-1. Within experimental error the dimer formation constant of the carboxy-terminal domain may be considered to be the same as that of the entire repressor whereas the isolated domain forms tetramers slightly less efficiently. It should be stressed that the potential error in K4 is higher than that in K2. The overall conclusion is that the two structural domains of LexA have also well-defined functional roles: the amino-terminal domain interacts with DNA and the carboxy-terminal domain is involved in dimerisation reinforcing in this way the binding of the LexA repressor to operator DNA.
Asunto(s)
Proteínas Bacterianas , Escherichia coli/análisis , Serina Endopeptidasas , Bacteriófago lambda/análisis , Sustancias Macromoleculares , Proteínas Represoras , UltracentrifugaciónRESUMEN
The effects of several hormones on intestinal brush border membrane enzymatic activities have been investigated in intestinal explants taken from the amphibian midwife toad at different developmental stages. Explants were treated for at least 2 days with thyroxine (0.1 microgram/ml of culture medium) or for 2 days with cortisol (25 micrograms/ml) or insulin (6 mU/ml). The hydrolases examined were maltase, trehalase, glucoamylase, and alkaline phosphatase. In the explants from tadpoles in prometamorphosis, thyroxine had no effect on hydrolase activities; cortisol increased the activity of only glucoamylase, and insulin increased activity of maltase, glucoamylase, and alkaline phosphatase. When the explants were taken from tadpoles at the beginning of climax, cortisol and insulin generally stimulated the enzyme activities studied. When taken from tadpoles at the end of climax, at the moment when the embryonic cells under the degenerating epithelium divide, cortisol and insulin had little effect on these activities. When the animals terminate their metamorphosis, the intestinal epithelium of the explants is totally newly formed (secondary epithelium). At this time, cortisol stimulated the activities of maltase, glucoamylase, and alkaline phosphatase, while insulin decreased the activities of maltase and glucoamylase.
Asunto(s)
Anuros/crecimiento & desarrollo , Hidrocortisona/farmacología , Hidrolasas/metabolismo , Insulina/farmacología , Intestinos/enzimología , Tiroxina/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Glucano 1,4-alfa-Glucosidasa/metabolismo , Intestinos/efectos de los fármacos , Microvellosidades/efectos de los fármacos , Microvellosidades/enzimología , Técnicas de Cultivo de Órganos , Trehalasa/metabolismo , alfa-Glucosidasas/metabolismoRESUMEN
In order to follow the fast kinetics of abortive initiation (lag time from 1 ms to 10 s), we have built a stopped-flow apparatus equipped for fluorescence detection. The small volume used for each assay (35 microliters), and the short dead time (approximately 0.5 ms) are the essential advantages of this apparatus. Supercoiling of DNA affects considerably the initiation of transcription from the uvrA promoter. It decreases the lag time due to the isomerisation process 3-fold. Nevertheless, it does not change significantly the product KBk2, which is indicative of promoter strength and shows that uvrA is an 'association-limited' promoter. The presence of the LexA repressor increases the lag time considerably. At least for small RNA polymerase concentrations this increase is stronger for supercoiled than for linearized DNA.
Asunto(s)
Proteínas Bacterianas/genética , ADN Bacteriano/metabolismo , ADN Superhelicoidal/metabolismo , Fluorometría/instrumentación , Regulación de la Expresión Génica , Plásmidos , Regiones Promotoras Genéticas , Serina Endopeptidasas , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Cinética , Proteínas Represoras/farmacología , Transcripción GenéticaRESUMEN
Ribosomal proteins from Escherichia coli MRE600 have been obtained by a new, mild purification procedure. This involves extraction of the subunits with salt followed by chromatographic fractionation in the presence of salt. The use of urea or other denaturing agents and conditions is avoided. A survey of the secondary structure of the 30 S and 50 S proteins, as observed by circular dichroic spectroscopy, is presented. The spectra have been analysed by a new procedure which uses a library of 16 circular dichroic spectra of proteins with a known three-dimensional structure. This method provides a more reliable analysis, especially of the contribution from beta-sheet. The results show that most of the 30 S proteins have a high alpha-helix content, whereas the 50 S proteins are more diverse. The latter group shows a larger contribution from beta-sheet. The data presented here are compared with those already published for a number of proteins which were, with one exception, prepared in the presence of urea. In most cases we find higher alpha-helix and beta-sheet values for the salt-extracted proteins than for the corresponding urea-treated proteins. In those cases, however, where special care was taken to renature the urea-treated proteins agreement is found to within the expected experimental error. The results show that salt-extracted ribosomal proteins have a well-defined secondary structure with a relatively small contribution from unordered structure.
Asunto(s)
Escherichia coli/metabolismo , Proteínas Ribosómicas , Dicroismo Circular , Conformación de Ácido Nucleico , Concentración Osmolar , Proteínas Ribosómicas/aislamiento & purificación , Sales (Química)RESUMEN
We show that an erroneous estimation of the quaternary structure of free protein distorts the quantitative analysis of its interaction with DNA, affecting especially the co-operativity value found. This could explain the discrepancy reported for the co-operativity value of the RecA-DNA interaction. The large cluster observed by electron microscopy indicates a very high co-operativity, whereas analysis of the binding isotherm indicates a moderate one, on the assumption of monomer. But if RecA is a large oligomer, the latter analysis would give a much higher co-operativity value and the former a smaller one, and they would be in accordance. Our sedimentation and light-scattering experiments suggest an oligomerization of about 30-mer or more, and support this explanation.
Asunto(s)
ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Rec A Recombinasas/metabolismo , Regulación Alostérica , Cinética , Sustancias Macromoleculares , Peso MolecularRESUMEN
It has been demonstrated recently by Poirier et al. (Poirier, G. G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C., and Mandel, P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3423-3427) that poly(ADP-ribosyl)ation of pancreatic nucleosomes causes relaxation of the chromatin superstructure through H1 modification. The in vitro effect of poly(ADP-ribose) synthesis and degradation on calf thymus chromatin was investigated by the time course incorporation of ADP-ribose, electron microscopy, analytical ultracentrifugation, and autoradiography of the protein acceptors. Purified calf thymus poly(ADP-ribose) polymerase and partially purified bull testis poly(ADP-ribose) glycohydrolase were used. Degradation of ADP-ribose units on hyper(ADP-ribosyl)ated H1 by poly(ADP-ribose) glycohydrolase restores the native condensed chromatin superstructure. This reversible conformational change induced by poly(ADP-ribosyl)ation on nucleosomal arrangement could be one of the mechanisms by which the accessibility of DNA polymerases and/or excision-repair enzymes is favored, the native structure being fully restorable.
Asunto(s)
Cromatina/ultraestructura , Azúcares de Nucleósido Difosfato/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Bovinos , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cinética , Nucleosomas/metabolismo , Poli Adenosina Difosfato Ribosa/biosíntesis , Timo/metabolismoRESUMEN
A rapid large-scale procedure for the purification of the LexA repressor of Escherichia coli is described. This procedure allows one to get more than 100 mg of purified protein from 100 g of bacterial paste with a purity of at least 97%. This method is comparable to earlier, far more complicated purification procedures giving clearly smaller yields. It is shown that the LexA protein may be identified spectroscopically by a large A235/A280 ratio and very pronounced ripples in the absorption spectrum arising from a high amount of phenylalanine residues with respect to that of the other aromatic amino acids. Polyacrylamide gel electrophoresis has been used to study the specific interaction of LexA with a recA operator fragment. The quaternary structure of LexA has been studied by equilibrium ultracentrifugation and sedimentation velocity measurements. The sedimentation coefficient increases with increasing LexA concentration, indicating that LexA is involved in self-association. This finding has been confirmed by equilibrium ultracentrifugation. The results are best described by a monomer-dimer and a subsequent dimer-tetramer equilibrium, with an association constant of 2.1 X 10(4) M-1 for the dimer and 7.7 X 10(4) M-1 for the tetramer formation. These relatively small association constants determined under near-physiological pH and salt conditions suggest that in vivo LexA should be essentially in the monomeric state. The degree to which LexA decreases the electrophoretic mobility of a 175 base pair fragment harboring the recA operator suggests that the recA operator interacts nevertheless with a LexA dimer. However, our results may be also explained by the binding of a LexA monomer with a simultaneous bending of the DNA fragment.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Proteínas Represoras/metabolismo , Serina Endopeptidasas , Factores de Transcripción/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Cinética , Sustancias Macromoleculares , Matemática , Peso Molecular , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
Isolated rat pancreatic polynucleosomes were poly(ADP-ribosylated) with purified calf thymus poly(ADP-ribose) polymerase. A time course study was performed using an NAD concentration of 200 microM and changes in nucleosomal structure were investigated by means of electron microscopy visualization and sedimentation velocity determinations. In parallel, analyses of histone H1 poly(ADP-ribosylation) and determinations of DNA polymerase alpha activity on ADP-ribosylated polynucleosomes were done at different time intervals. A direct kinetic correlation between ADP-ribose incorporation, polynucleosome relaxation amd histone H1 hyper-ADP-ribosylation was established. In addition, DNA polymerase alpha activity was highly stimulated on ADP-ribosylated polynucleosomes as compared to control ones, suggesting increased accessibility of DNA to enzymatic action. Because of the strong evidence implicating histone H1 in the maintenance of higher-ordered chromatin structures, the present study may provide a basis for the interpretation of the involvement of the histone H1 ADP-ribosylation reaction in DNA rearrangements during DNA repair, replication or gene expression.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Histonas/metabolismo , Azúcares de Nucleósido Difosfato/metabolismo , Nucleosomas/metabolismo , Animales , Bovinos , ADN Polimerasa II/metabolismo , Cinética , Microscopía Electrónica , Páncreas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ratas , UltracentrifugaciónRESUMEN
A chemical quenched-flow apparatus is described which measures, in a unique stroke, enough data points (8-11) for establishing the kinetics curve of a reaction. Only very small volumes of reaction solutions (2 X 500 microliters) are required. The time intervals between which the kinetic data may be measured range from 5 to 37 ms and from 120 to 450 ms with the corresponding mixing times of 0.6 and 5 ms, respectively. This apparatus was used to investigate the pre-steady-state domain of the aminoacylation reaction of tRNAVal by valyl-tRNA synthetase from yeast.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Cinética , Reología , Valina-ARNt Ligasa/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
The hexanucleoside pentaphosphate d(m5CpGpm5CpGpm5CpG) has been studied in solution by ultra-violet absorption, circular dichroism and 31P nuclear magnetic resonance under various experimental conditions. In 0.2 M NaClO4 at low temperature, an hexamer duplex is formed which has a B or B-like conformation. As the salt concentration is increased, a transition from a B-form to the Z-form occurs and is complete in 3 M NaClO4. In 3 M NaClO4, the behavior of the Z double helix is complex as a function of temperature. The variation of the circular dichroism at 295 nm is biphasic. A first transition occurs over a large range of temperature and corresponds to a conformational change due to a non-cooperative intramolecular process. Ultra-violet absorption and 31P nuclear magnetic resonance show that the new conformation arising from a distortion of the backbone is not similar to that observed in low salt conditions (B-form). At high hexanucleotide concentration, aggregates are formed. The second transition is cooperative and corresponds to the melting of a double stranded helix into single strands.
Asunto(s)
Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos , Oligonucleótidos , Dicroismo Circular , Estabilidad de Medicamentos , Cinética , Espectroscopía de Resonancia Magnética , Concentración Osmolar , Espectrofotometría Ultravioleta , TemperaturaRESUMEN
The organotypic culture of the gastric region is carried out on premetamorphic Discoglossus pictus larvae. Adding thyroxine to the culture medium provokes various transformations. On the cytological level, the reactions observed, which are variable depending on the cell category concerned, can be divided into two types of phenomena: histolytic and histogenetic. Autophagia linked to lysosome intervention is frequently found among the histolytic processes. Autophagic vacuoles and residual bodies are observed. The gastric lumen is filled with deteriorated cells that probably come from the degeneration of the tadpole epithelium (primary epithelium). The incorporation of tritiated thymidine makes it possible to study the evolution of cell proliferation in the control and in the thyroxinated cultures. After a 1-2 day latency period, possibly due to the adjustment of the tissue to the culture environment, the incorporation of the radioprecursor H3-thymidine into the epithelium and the tunica muscularis of thyroxine-treated gut tissue increased on day 3, reached a maximum on day 5, and then dropped slightly on day 7. In the control cultures H3-thymidine incorporation showed the same pattern but lower levels on the same days. The histolytic phenomena induced by thyroxine in vitro are comparable to those of natural metamorphosis. On the other hand, the histogenetic phenomena are incomplete. Proliferating and transitional phases occur but neoformated (or secondary) epithelium does not replace the degenerated primary epithelium, whatever the culture time.
Asunto(s)
Estómago/citología , Tiroxina/farmacología , Animales , Anuros , Larva/crecimiento & desarrollo , Técnicas de Cultivo de Órganos , Estómago/efectos de los fármacos , Factores de TiempoRESUMEN
Core particle DNA unfolding and refolding are followed by stopped-flow circular dichroism technique. When core particles are dissociated in the stopped-flow cuvette, the high CD deviation corresponding to the dissociated state is reached in the first millisecond, which means that the dissociation process is completed within the dead time of the apparatus which is approximately 1 ms. The same conclusion can be drawn when core particles are reassociated, since the low CD value, typical of the associated state, is immediately reached. Similarly histone release from chromatin is a very fast process. We also include some points of discussion about core particle assembly process.
Asunto(s)
Cromatina/metabolismo , ADN/metabolismo , Histonas/metabolismo , Animales , Dicroismo Circular , Cinética , Conformación de Ácido Nucleico , Desnaturalización Proteica , Factores de TiempoRESUMEN
In avian erythroid cells the erythrocyte-specific histone H5 is involved, like H1, in the packing of nucleosomes in the 25-nm chromatin fibers. In this study the distribution of histone H5 along the polynucleosomal chains was visualized by immunoelectron microscopy. Trinucleosomes from chicken erythrocytes and liver were used in order to test the specificity of the reaction with purified rabbit anti-H5 antibodies at various ionic strengths (5-80 mM). Long-chain chromatin was then reacted with anti-H5 antibodies and with sorted monomeric ferritin conjugate under chosen conditions. The antigenic determinants of histone H5 in the 25-nm fiber of long-chain chromatin (at 80 mM NaCl) are as accessible to the specific antibodies as in trinucleosomes. When the immunocomplexes were examined by electron microscopy in a low-ionic-strength buffer, permitting maximum extension of the chromatin structure on the grid, clusters of compacted nucleosomes were seen, separated by short regions of relaxed nucleosomes. Single nucleosomes enlarged by the antibodies are sometimes visible in the extended domains. We conclude that histone H5 is located primarily on series of adjacent nucleosomes but it can also be found on single nucleosomes located in the H1-enriched extended domains.
Asunto(s)
Cromatina/análisis , Histonas/análisis , Animales , Núcleo Celular/ultraestructura , Pollos , Cromatina/ultraestructura , Eritrocitos/ultraestructura , Histocitoquímica , Microscopía Electrónica/métodosRESUMEN
Cyanophora paradoxa, a unicellular flagellate, contains cyanelles which are supposed to be cyanobacterial origin. DNA was isolated from subcellular fractions and separated according to density components in CsC1 density gradients. The main DNA component, comprising more than 90% of the total DNA, has a buoyant density of 1.724 g X cm-3. Several subsfractions in the range from 1.718 g X cm-3 to 1.735 g X cm-3 are contained in this component. This DNA of high complexity was considered to be host nuclear DNA. The DNA from the endosymbiotic cyanelles, which were isolated, treated with DNase, and purified by sucrose density gradient centrifugation exhibited a buoyant density of 1.692 g X cm-3 in one strain and 1.695 g X cm-3 in a second strain. Both cyanelle DNAs (cyDNA) have a complexity of approximately 126 X 10(3) base pairs and comprise about 5% of the total cellular DNA content. Two additional DNA components of low complexity were isolated from crude cyanelle pellets obtained without DNase treatment. The larger of these, approximately 48 X 10(3) base pairs in size, had a density of approximately 1.688 g X cm-3. The second component, about 15 X 10(3) base pairs in size, banded in the density range between 1.710 g X cm-3 and 1.720 g X cm-3. The latter is associated with nuclear DNA. The 48 X 10(3)-base-pair component was located in the cytosol and could be obtained after CsC1/ethidium bromide density gradient centrifugation at the position of covalently closed circular DNA. Both these components amounted to approximately 0.5-1% of total DNA. A further DNA component with a complexity of more than 150 X 10(3) base pairs, enriched in fractions where mitochondria are expected, was not characterized further. The density was intermediate between cyDNA and nuclear DNA (1.710-1.720 g X cm-3) and it amounted to 1-2% of the total DNA. Our results indicate that the DNA from cyanelles, believed to be endosymbiotic cyanobacteria, is not more complex than higher plant chloroplast DNAs.
Asunto(s)
Cianobacterias/metabolismo , ADN/aislamiento & purificación , Centrifugación por Gradiente de Densidad , Fenómenos Químicos , Química , Cianobacterias/ultraestructura , SimbiosisRESUMEN
An apparently homogeneous population of core particles is in fact composed of three subpopulations which behave differently when exposed to a high concentration of ethidium bromide or to 0.6 M NaCl. These subspecies have been identified by the use of several techniques, viz., electron microscopy, sedimentation velocity and circular dichroism. The electrophoretic analysis of their DNA leads to the conclusion that core particle stability critically depends upon a small number of terminal nucleotides.
Asunto(s)
Cromatina/ultraestructura , ADN/sangre , Eritrocitos/análisis , Animales , Pollos , Estabilidad de Medicamentos , Etidio , Microscopía Electrónica , Concentración Osmolar , Cloruro de SodioRESUMEN
We determined 90% of the primary structure of E.coli MRE 600 23S rRNA by applying the sequencing gel technique to products of T1, S1, A and Naja oxiana nuclease digestion. Eight cistron heterogeneities were detected, as well as 16 differences with the published sequence of a 23S rRNA gene of an E.coli K12 strain. The positions of 13 post-transcriptionally modified nucleotides and of single-stranded, double-stranded and subunit surface regions of E.coli 23S rRNA were identified. Using these experimental results and by comparing the sequences of E.coli 23S rRNA, maize chloro. 23S rRNA and mouse and human mit 16S rRNAs, we built models of secondary structure for the two 23S rRNAs and for large portions of the two mit rRNAs. The structures proposed for maize chloroplast and E.coli 23S rRNAs are very similar, consisting of 7 domains closed by long-range base-pairings. In the mitochondrial 16S rRNAs, 3 of these domains are strongly reduced in size and have a very different primary structure compared to those of the 23S rRNAs. These domains were previously found to constitute a compact area in the E.coli 50S subunits. The conserved domains do not belong to this area and contain almost all the modified nucleotides. The most highly conserved domain, 2042-2625, is probably part of the ribosomal A site. Finally, our study strongly suggests that in cytoplasmic ribosomes the 3'-end of 5.8S rRNA is basepaired with the 5'-end of 26S rRNA. This confirms the idea that 5.8S RNA is the counterpart of the 5'-terminal region of prokaryotic 23S rRNA.
Asunto(s)
Cloroplastos/análisis , Escherichia coli/análisis , Mitocondrias/análisis , Conformación de Ácido Nucleico , ARN Bacteriano , ARN Ribosómico , Animales , Secuencia de Bases , Humanos , Ratones , Modelos Moleculares , Zea maysRESUMEN
The respiratory protein, erythrocruorin, of the annelid Arenicola marina was investigated. Spectral properties show many analogies with those of vertebrate hemoglobine. 144 heme groups (ferroprotoporphyrin) were found in the whole molecule, which has a relative molecular mass of 3.56 X 10(6), as determined by sedimentation equilibrium, and an isoelectric point of 4.69. Protein dissociation patterns were analysed by electrophoresis after denaturation in the presence of dodecylsulfate, with and without 2-mercaptoethanol. A tentative model associating molecular mass of the native molecule, heme content, molecular mass of the polypeptide chains and functional properties is proposed. A twelfth subunit of A. marina erythrocruorin would contain twelve heme groups arranged in three functional units made up of four protomers, half of these being covalently bound to non-heme chains; two structural chains would be spatially arranged as bonds between the subunits.
Asunto(s)
Eritrocruorinas , Hemoglobinas , Poliquetos/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Hemo/análisis , Focalización Isoeléctrica , Peso Molecular , Dodecil Sulfato de Sodio , Espectrofotometría , UltracentrifugaciónRESUMEN
Core particles were prepared from mature chicken erythrocytes chromatin, according to the method of Lutter (J. Mol. Biol. 124, 391, 1978) with one major modification: after the second digestion, zonal centrifugation was used to isolate the core particle, instead of chromatography on Sepharose 6B. By using circular dichroism and electron microscopy, we were able to follow each step of the preparation and to offer an explanation of the discrepancies found in previous preparations and in our own preparations.
Asunto(s)
Cromatina/aislamiento & purificación , Animales , Pollos , Dicroismo Circular , ADN/sangre , Eritrocitos/metabolismo , Eritrocitos/ultraestructura , Histonas/sangre , Métodos , Microscopía Electrónica , Conformación de Ácido NucleicoRESUMEN
Ethidium bromide intercalation into DNA of nuclease digested erythrocyte chromatin and core particle, was followed at low ionic strength by fluorescence measurements, equilibrium dialysis using 14C labelled dye, circular dichroism and electron microscopy. High affinity binding sites in the chromatin are no more present in the core particle, i.e. when the linker is removed. In the case of core particle, a cooperative process occurs, accompanied by a partial stripping of the DNA from the core histone. Finally two populations of core particles can be detected by electron microscopy as far as their binding properties are concerned.
Asunto(s)
Cromatina , ADN , Etidio , Animales , Fenómenos Químicos , Química , Pollos , Dicroismo Circular , ADN/sangre , Eritrocitos/análisis , Cinética , Conformación de Ácido Nucleico , Espectrometría de FluorescenciaRESUMEN
Yeast valyl-tRNA synthetase and its complexes with yeast tRNAVal were investigated by means of analytical ultracentrifugation. A molecular weight of 125 700 +/- 1500 and a sedimentation coefficient (SO 20, w) of 6.3 +/- 0.3 were found for the native enzyme. When the enzyme (3--60 muM) was mixed with its cognate tRNA, several types of complex were observed, depending on the relative amounts of the two macromolecules. In the presence of equimolecular amounts of tRNA and enzyme, a complex formed by the association of one of each molecule was observed with a sedimentation coefficient of about 7.3 S. However, for tRNA/enzyme stoichiometries lower than one, beside the 1 : 1 complex, a complex of higher molecular weight was observed, with a sedimentation coefficient of about 10.0 S which fits with the association of two valyl-tRNA synthetase molecules with one tRNA molecule. This 2 : 1 complex was predominant from tRNA/enzyme stoichiometries lower than 0.3. It dissociated into the 1 : 1 complex upon addition of monovalent salts or MgCl2, suggesting the electrostatic nature of the interaction in this association. All these association and dissociation phenomena were detected over a large range of pH (6.0--7.5) and in various buffers.