RESUMEN
The sequences of genes encoding homologues of 1-aminocyclopropane-1-carboxylate (ACC) synthase, the first enzyme in the two-step biosynthetic pathway of the important plant hormone ethylene, have recently been found in Fugu rubripes and Homo sapiens (Peixoto et al., Gene 246 (2000) 275). ACC synthase (ACS) catalyzes the formation of ACC from S-adenosyl-L-methionine. ACC is oxidized to ethylene in the second and final step of ethylene biosynthesis. Profound physiological questions would be raised if it could be demonstrated that ACC is formed in animals, because there is no known function for ethylene in these organisms. We describe the cloning of the putative human ACS (PHACS) cDNA that encodes a 501 amino acid protein that exhibits 58% sequence identity to the putative Fugu ACS and approximately 30% sequence identity to plant ACSs. Purified recombinant PHACS, expressed in Pichia pastoris, contains bound pyridoxal-5'-phosphate (PLP), but does not catalyze the synthesis of ACC. PHACS does, however, catalyze the deamination of L-vinylglycine, a known side-reaction of apple ACS. Bioinformatic analysis indicates that PHACS is a member of the alpha-family of PLP-dependent enzymes. Molecular modeling data illustrate that the conservation of residues between PHACS and the plant ACSs is dispersed throughout its structure and that two active site residues that are important for ACS activity in plants are not conserved in PHACS.
Asunto(s)
ADN Complementario/genética , Liasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/química , Frutas/enzimología , Genes/genética , Humanos , Liasas/química , Liasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Plantas/enzimología , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The yeast Candida glabrata rapidly autoactivates transcription of the AMT1 gene in response to potentially toxic copper levels through the copper-inducible binding of the Amt1 transcription factor to a metal response element (MRE) within a positioned nucleosome. Our previous studies have characterized the role of a 16 bp homopolymeric dA:dT DNA structural element in facilitating rapid Amt1 access to the AMT1 promoter nucleosomal MRE. In this study, we have used the genetically more facile yeast Saccharomyces cerevisiae to identify additional cellular factors that are important for promoting rapid autoactivation of the AMT1 gene in response to toxic copper levels. We demonstrate that the Swi/Snf nucleosome remodelling complex and the histone acetyltransferase Gcn5 are both essential for AMT1 gene autoregulation, and that the requirement for these chromatin remodelling factors is target gene specific. Chromatin accessibility measurements performed in vitro and in vivo indicate that part of the absolute requirement for these factors is derived from their involvement in facilitating nucleosomal access to the AMT1 promoter MRE. Additionally, these data implicate the involvement of Swi/Snf and Gcn5 at multiple levels of AMT1 gene autoregulation.
Asunto(s)
Candida/metabolismo , Cobre/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Proteínas Fúngicas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas de Unión al ARN , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/metabolismo , Acetilación , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Biodegradación Ambiental , Candida/genética , Proteínas Portadoras , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Histona Acetiltransferasas , Histonas/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Proteínas Quinasas/genética , Ribonucleoproteína Nuclear Pequeña U1/genética , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Endodontics as a discipline has offered patients the opportunity to maintain their natural teeth. As the population expands and ages, the demand for endodontic therapy can be expected to increase as patients seek dental options to keep their teeth for a lifetime. CLINICAL IMPLICATIONS: New materials, techniques and instruments are entering the market-place to assist dentists in providing patients with more predictable and reliable endodontic treatment. In addition, these new systems make the delivery of endodontic services more efficient. This article describes these advances in endodontic treatment for dentists interested in incorporating these advances into their clinical practice.
Asunto(s)
Tratamiento del Conducto Radicular/tendencias , Compuestos de Aluminio/química , Compuestos de Calcio/química , Aleaciones Dentales/química , Equipo Dental de Alta Velocidad , Combinación de Medicamentos , Endoscopía , Diseño de Equipo , Predicción , Humanos , Microscopía , Níquel/química , Óxidos/química , Reproducibilidad de los Resultados , Materiales de Obturación del Conducto Radicular/química , Irrigantes del Conducto Radicular/uso terapéutico , Obturación del Conducto Radicular/instrumentación , Obturación del Conducto Radicular/métodos , Tratamiento del Conducto Radicular/instrumentación , Tratamiento del Conducto Radicular/métodos , Silicatos/química , Tecnología Odontológica , Titanio/química , TorqueRESUMEN
Eukaryotic organisms ranging from yeast to humans maintain a large amount of genetic information in the highly compact folds of chromatin, which poses a large DNA accessibility barrier to rapid changes in gene expression. The ability of the yeast Candida glabrata to survive copper insult requires rapid transcriptional autoactivation of the AMT1 copper-metalloregulatory transcription factor gene. The kinetics of AMT1 autoactivation is greatly enhanced by homopolymeric (dA-dT) element (A16)-mediated nucleosomal accessibility for Amt1p to a metal response element in this promoter. Analysis of the nucleosomal positional requirements for the A16 element reveal an impaired ability of the A16 element to stimulate AMT1 autoregulation when positioned downstream of the metal response element within the nucleosome, implicating an inherent asymmetry to the nucleosome positioned within the AMT1 promoter. Importantly, we demonstrate that the A16 element functions to enhance nucleosomal access and hormone-stimulated transcriptional activation for the mammalian glucocorticoid receptor, in a rotational phase-dependent manner. These data provide compelling evidence that nucleosomal homopolymeric (dA-dT) elements provide enhanced DNA access to diverse classes of transcription factors and suggest that these elements may function in this manner to elicit rapid transcriptional responses in higher eukaryotic organisms.
Asunto(s)
Proteínas de Unión al ADN/genética , Nucleosomas/fisiología , Poli dA-dT/química , Factores de Transcripción/genética , Activación Transcripcional , Animales , Candida/genética , Proteínas Fúngicas , Regiones Promotoras Genéticas , Ratas , Receptores de Glucocorticoides/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Gene expression in response to heat shock is mediated by the heat shock transcription factor (HSF), which in yeast harbors both amino- and carboxyl-terminal transcriptional activation domains. Yeast cells bearing a truncated form of HSF in which the carboxyl-terminal transcriptional activation domain has been deleted [HSF(1-583)] are temperature sensitive for growth at 37 degreesC, demonstrating a requirement for this domain for sustained viability during thermal stress. Here we demonstrate that HSF(1-583) cells undergo reversible cell cycle arrest at 37 degreesC in the G2/M phase of the cell cycle and exhibit marked reduction in levels of the molecular chaperone Hsp90. As in higher eukaryotes, yeast possesses two nearly identical isoforms of Hsp90: one constitutively expressed and one highly heat inducible. When expressed at physiological levels in HSF(1-583) cells, the inducible Hsp90 isoform encoded by HSP82 more efficiently suppressed the temperature sensitivity of this strain than the constitutively expressed gene HSC82, suggesting that different functional roles may exist for these chaperones. Consistent with a defect in Hsp90 production, HSF(1-583) cells also exhibited hypersensitivity to the Hsp90-binding ansamycin antibiotic geldanamycin. Depletion of Hsp90 from yeast cells wild type for HSF results in cell cycle arrest in both G1/S and G2/M phases, suggesting a complex requirement for chaperone function in mitotic division during stress.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Activación Transcripcional , Antibióticos Antineoplásicos/farmacología , Benzoquinonas , Ciclo Celular , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas HSP90 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico , Lactamas Macrocíclicas , Quinonas/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Relación Estructura-Actividad , Temperatura , Factores de Transcripción/fisiologíaRESUMEN
The essential yet toxic nature of copper demands tight regulation of the copper homeostatic machinery to ensure that sufficient copper is present in the cell to drive essential biochemical processes yet prevent the accumulation to toxic levels. In Saccharomyces cerevisiae, the nutritional copper sensor Mac1p regulates the copper-dependent expression of the high affinity Cu(I) uptake genes CTR1, CTR3, and FRE1, while the toxic copper sensor Ace1p regulates the transcriptional activation of the detoxification genes CUP1, CRS5, and SOD1 in response to copper. In this study, we characterized the tandem regulation of the copper uptake and detoxification pathways in response to the chronic presence of elevated concentrations of copper ions in the growth medium. Upon addition of CuSO4, mRNA levels of CTR3 were rapidly reduced to eightfold the original basal level whereas the Ace1p-mediated transcriptional activation of CUP1 was rapid and potent but transient. CUP1 expression driven by an Ace1p DNA binding domain-herpes simplex virus VP16 transactivation domain fusion was also transient, demonstrating that this mode of regulation occurs via modulation of the Ace1p copper-activated DNA binding domain. In vivo dimethyl sulfate footprinting analysis of the CUP1 promoter demonstrated transient occupation of the metal response elements by Ace1p which paralleled CUP1 mRNA expression. Analysis of a Mac1p mutant, refractile for copper-dependent repression of the Cu(I) transport genes, showed an aberrant pattern of CUP1 expression and copper sensitivity. These studies (i) demonstrate that the nutritional and toxic copper metalloregulatory transcription factors Mac1p and Ace1p must sense and respond to copper ions in a dynamic fashion to appropriately regulate copper ion homeostasis and (ii) establish the requirement for a wild-type Mac1p for survival in the presence of toxic copper levels.
Asunto(s)
Proteínas de Transporte de Catión , Cobre/metabolismo , FMN Reductasa , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Antiportadores/metabolismo , Sitios de Unión , Transporte Biológico , Proteínas Portadoras , Cationes/metabolismo , Transportador de Cobre 1 , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Inactivación Metabólica , Proteínas de la Membrana/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , Pruebas de Sensibilidad Microbiana , NADH NADPH Oxidorreductasas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas SLC31 , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Listening, teaching, understanding, exploring, explaining: these are the foundations of a sound patient-physician relationship. From these skills, we can then proceed to discussions on difficult topics such as preferences for end-of-life care. We can share bad news without destroying hope. We can show what makes the medical profession unlike any other. This issue of The Journal addresses the handling of medical errors, the termination of mechanical ventilatory support, ethical problems in managed care, and confidentiality issues in the computer era. Guidelines for institutional ethics committees also are presented. These are only a sampling of topics that cut to the heart of bioethics, patient communication, and contemporary medical practice. The more that we study such issues, the more we understand the contributions of medical ethics to medical practice, and the better we serve our patients.
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Bioética , Comunicación , Relaciones Médico-Paciente , Confidencialidad , Toma de Decisiones , Ética Médica , Recursos en Salud , Humanos , Programas Controlados de Atención en Salud , Inutilidad Médica , Ciencia del Laboratorio Clínico , Educación del Paciente como Asunto , Enseñanza , Cuidado TerminalAsunto(s)
Ética Médica , Programas Controlados de Atención en Salud , Relaciones Médico-Paciente , Libertad , Salud , Costos de la Atención en Salud , Asignación de Recursos para la Atención de Salud , Humanos , Evaluación de Resultado en la Atención de Salud , Defensa del Paciente , Formulación de Políticas , Evaluación de Procesos, Atención de Salud , Calidad de la Atención de Salud , Responsabilidad Social , Valores SocialesRESUMEN
Copper is required for many biological processes but is toxic at high cellular concentrations, so levels in the cell must be strictly controlled. Copper-binding motifs have been identified and characterized in many proteins. The way in which copper is coordinated by these motifs is important for the transport and distribution of intracellular copper and for the effective functioning of copper-dependent enzymes.
Asunto(s)
Cobre/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Transporte Biológico Activo , Proteínas Portadoras/metabolismo , Catálisis , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Cobre/toxicidad , ADN/genética , ADN/metabolismo , Degeneración Hepatolenticular/metabolismo , Humanos , Inactivación Metabólica , Síndrome del Pelo Ensortijado/metabolismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Modelos Moleculares , Conformación Proteica , Transducción de Señal , Distribución TisularRESUMEN
Rapid transcriptional autoactivation of the Candida glabrata AMT1 copper metalloregulatory transcription factor gene is essential for survival in the presence of high extracellular copper concentrations. Analysis of the interactions between purified recombinant AMT1 protein and the AMT1 promoter metal regulatory element was carried out by a combination of missing-nucleoside analysis, ethylation interference, site-directed mutagenesis, and quantitative in vitro DNA binding studies. The results of these experiments demonstrate that monomeric AMT1 binds the metal regulatory element with very high affinity and utilizes critical contacts in both the major and minor grooves. A single adenosine residue in the minor groove, conserved in all known yeast Cu metalloregulatory transcription factor DNA binding sites, plays a critical role in both AMT1 DNA binding in vitro and Cu-responsive AMT1 gene transcription in vivo. Furthermore, a mutation in the AMT1 Cu-activated DNA binding domain which converts a single arginine, found in a conserved minor groove binding domain, to lysine markedly reduces AMT1 DNA binding affinity in vitro and results in a severe defect in the ability of C. glabrata cells to mount a protective response against Cu toxicity.
Asunto(s)
Candida/genética , Cobre/farmacología , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Candida/efectos de los fármacos , Secuencia Conservada , Proteínas de Unión al ADN/genética , Proteínas Fúngicas , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
This article proposes that the medical community has a responsibility to guide and to treat patients through death just as it guides and treats them through life. A number of misunderstood concepts relating to this responsibility are discussed. An approach to the management of death that requires an embracing of medical uncertainty is developed.
Asunto(s)
Actitud Frente a la Muerte , Eutanasia , Terminología como Asunto , Incertidumbre , Toma de Decisiones , Principio del Doble Efecto , Ética , Eutanasia/legislación & jurisprudencia , Humanos , Intención , Defensa del Paciente , Responsabilidad Social , Estados Unidos , Privación de TratamientoRESUMEN
The universal presumption of consent for cardiopulmonary resuscitation creates several practical and ethical dilemmas and should be challenged. Ethically based decision making demands a reality-based dialogue about resuscitation with patients and the community at large.
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Ética Médica , Consentimiento Informado , Resucitación , Beneficencia , Revelación , Servicios Médicos de Urgencia/normas , Humanos , Autonomía Personal , Relaciones Profesional-Paciente , Asignación de Recursos , Resucitación/educación , Resucitación/normas , Órdenes de Resucitación , Estados Unidos , Privación de TratamientoRESUMEN
The current standards for surrogate decision making for children are analyzed in this article. A number of potential pitfalls are involved with this issue, including questions about the child's maturity and competence. The present approach for medical decision making for noncompetent children is the "best interest" standard. In many medical decisions, the "best interest" standard is not applicable and a "rational parent" standard is presented.
Asunto(s)
Cuidados Críticos , Ética Médica , Cuidado Intensivo Neonatal , Pediatría , Niño , Preescolar , Cuidados Críticos/legislación & jurisprudencia , Toma de Decisiones , Regulación Gubernamental , Humanos , Lactante , Recién Nacido , Cuidado Intensivo Neonatal/legislación & jurisprudencia , Tutores Legales , Menores , Consentimiento Paterno , Paternalismo , Pediatría/legislación & jurisprudencia , Autonomía Personal , Medición de Riesgo , Valores Sociales , Estrés Psicológico , Obtención de Tejidos y Órganos , Estados UnidosRESUMEN
In vitro temperatures produced in the root canal and on the root surface were measured simultaneously as heated gutta-percha was injected into the prepared canal. The canals were obturated with the Obtura II heated gutta-percha system with temperature settings of 160, 185, and 200 degrees C. The mean intracanal temperatures ranged from 40.21 to 57.24 degrees C, whereas the mean root surface temperatures were recorded from 37.22 to 41.90 degrees C for all three temperatures tested. The rise in temperature on the root surface was below the critical level of 10 degrees C and should not cause damage to the periodontal ligament.
Asunto(s)
Temperatura Corporal , Cavidad Pulpar/fisiología , Gutapercha , Obturación del Conducto Radicular/métodos , Raíz del Diente/fisiología , Calor , Humanos , Ligamento Periodontal/lesiones , Obturación del Conducto Radicular/efectos adversos , Propiedades de SuperficieRESUMEN
A 76-year-old white woman with ovarian adenocarcinoma developed massive biliptysis following an unexpected respiratory arrest. The possibility of active right upper quadrant disease had not been entertained until she developed this complication of a biliobronchial fistula. Because this sign had not previously been observed by any of us, it led to a comprehensive review of the topic of biliobronchial fistula.
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Fístula Biliar/complicaciones , Fístula Bronquial/complicaciones , Anciano , Bilis/química , Fístula Biliar/diagnóstico , Fístula Biliar/etiología , Fístula Bronquial/diagnóstico , Fístula Bronquial/etiología , Colestasis Extrahepática/complicaciones , Colestasis Extrahepática/patología , Conducto Colédoco/patología , Resultado Fatal , Femenino , Humanos , Síndrome de Dificultad Respiratoria/etiología , Esputo/químicaRESUMEN
In vitro intracanal temperatures produced by the injection of high-temperature thermoplasticized gutta-percha were measured. Obturations of a standard root canal were performed using an Obtura II heated gutta-percha system at temperature settings of 160, 185, and 200 degrees C. There was an increase in the recorded temperatures in the root canal for all three temperature settings with the mean temperature range from 38.52 degrees C to 61.58 degrees C. The mean temperature of the empty heating chamber was also measured and was accurately indicated on the control unit. However, the mean temperature of the gutta-percha in the heating chamber was almost 20 degrees C cooler than expected at each of the three settings. Finally, there was more than a 100 degrees C decrease in the mean temperature of the extruded gutta-percha in relation to the temperature of the gutta-percha in the heating chamber. Several clinically relevant observations were noted. Each of these observations was related to the temperature setting of the Obtura II control unit.
Asunto(s)
Cavidad Pulpar/fisiopatología , Gutapercha , Obturación del Conducto Radicular/métodos , Temperatura Corporal , Calor , HumanosRESUMEN
OBJECTIVE: To empirically describe changes in terminal care management behavior over time with the advent of natural death acts and public dialogue and institutional policy regarding terminal care. DESIGN: Retrospective analysis of medical decision-making and outcome was performed in a cohort of 237 intensive care unit (ICU) patients who received a do-not-resuscitate decision. SETTING: Medical ICU in a tertiary care center. PATIENTS: The cohort of 237 consecutive patients who received a terminal care decision in the ICU, i.e., a do-not-resuscitate decision with or without additional limitation of care, represented 9.3% of 2,185 patients admitted to the ICU over a 4-yr period. Brain-dead patients were excluded from the cohort. INTERVENTIONS: Implementation of hospital-wide policies on do-not-resuscitate decisions and discontinuation of life-prolonging procedures in 1986. MEASUREMENTS AND MAIN RESULTS: A change in frequency and nature of terminal care decisions occurred. By 1988, do-not-resuscitate decisions occurred twice as often as in 1984 (p = .016) compared with ICU deaths. Formal terminal wean decisions, i.e., additional limitation or withdrawal of care, occurred more frequently after 1985 (p = .027). The hospital mortality rate for the do-not-resuscitate cohort was 96.4% (226/237). The diagnosis of cardiac arrest was correlated with subsequent terminal care decisions (p = .0005, r2 = .08). Age of >56 yrs was increasingly correlated with probability of a terminal care decision (p < .0001, r2 = .05). White women received withdrawal of care most frequently, followed by white men, African American men, and African American women. Outcomes analysis indicated that after a do-not-resuscitate decision, most nonsurvivors died within 48 hrs. Eleven patients without additional limitation or withdrawal of care survived to hospital discharge (11/237 [4.6%]). No patient survived a terminal wean. CONCLUSIONS: There is now an increasing probability that impending death will be acknowledged by a formal terminal care decision. Such decisions may become even more frequent with the dialogue generated by the Patient Self Determination Act and the advent of decisions based on physiologic futility.
Asunto(s)
Unidades de Cuidados Intensivos , Órdenes de Resucitación , Cambio Social , Cuidado Terminal/tendencias , Privación de Tratamiento , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Toma de Decisiones , Investigación Empírica , Eutanasia Pasiva , Femenino , Mortalidad Hospitalaria , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de TiempoRESUMEN
BACKGROUND: In effect since 1991, the Patient Self-Determination Act (PSDA) requires that institutions receiving government insurance payments document that they have informed patients of their right to decide on life-preserving measures. Implementing the PSDA should make discussion of advance directives a routine part of acute care hospital admissions. Yet the proportion of those actually completing advance directives such as living wills remains relatively small. METHODS: A telephone survey questionnaire was administered to patients who were hospitalized before and after the implementation of the PSDA. Survey questions probed patient knowledge about living wills and behavior toward obtaining living wills. RESULTS: Patient knowledge about advance medical directives correlated positively with race (white), income (> or = $10K), and level of education (high school or more). Moreover, a significantly greater number of patients hospitalized after implementation of the PSDA knew about living wills than the number of those hospitalized before the Act's implementation. However, actually obtaining a living will correlated positively with age (> 36 years) alone, and implementation of the PSDA was not related to the number of patients who obtained a living will. CONCLUSIONS: Although the study results show that the measures the hospital in the study used to meet PSDA requirements increased patient awareness of living wills, they failed to increase the number of patients who act on this awareness. This finding indicates that simply informing patients about their right of self-determination is insufficient to meet the intended goals of the legislation.
Asunto(s)
Directivas Anticipadas/legislación & jurisprudencia , Difusión de la Información , Participación del Paciente/legislación & jurisprudencia , Adulto , Femenino , Libertad , Conocimientos, Actitudes y Práctica en Salud , Humanos , Voluntad en Vida/legislación & jurisprudencia , Masculino , Persona de Mediana Edad , Cooperación del Paciente , Educación del Paciente como Asunto , Estados UnidosRESUMEN
tRNA-guanine transglycosylase (TGT) is the enzyme responsible for the post-transcriptional modification of specific tRNAs (those for Asn, Asp, His, and Tyr) with the hypermodified base, queuine. In Escherichia coli this enzyme catalyzes the exchange of guanine-34 in the anticodon with preQ1, which is subsequently further modified to queuine. There is evidence that such hypermodified tRNA molecules may play a role in the control of cell proliferation and differentiation. In order to perform detailed, in vitro mechanistic studies and to probe the tRNA-enzyme interaction, we have generated unmodified E. coli tRNA(Tyr) and truncated analogues using an in vitro RNA synthesis system suggested by Milligan and Uhlenbeck [Milligan, J. F., & Uhlenbeck, O. C. (1989) Methods Enzymol. 180, 51-62]. From this system we have generated three tRNA analogues totally devoid of any post-transcriptional modifications. In order to compare the unmodified tRNA with the true physiological substrate for TGT, that is, tRNA that contains all modified bases except queuine, we have isolated E. coli tRNA(Tyr) from an overexpressing clone in a TGT-deficient strain of E. coli. We report here that unmodified, full-length tRNA(Tyr) serves as a substrate for TGT with kinetic parameters that are, within experimental error, the same as those for in vivo isolated tRNA(Tyr). This indicates that other post-transcriptional modifications have negligible effects upon TGT recognition of tRNA. A 17-base oligoribonucleotide, corresponding to the anticodon loop and stem, is also a substrate for TGT with only a 20-fold loss in Vmax/KM, versus the full-length tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Escherichia coli/enzimología , Pentosiltransferasa/metabolismo , ARN de Transferencia de Tirosina/química , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Geobacillus stearothermophilus/genética , Cinética , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Reacción en Cadena de la Polimerasa , ARN de Transferencia de Tirosina/genética , ARN de Transferencia de Tirosina/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
tRNA-guanine transglycosylase (TGT) is the enzyme responsible for the posttranscriptional modification of specific tRNAs (Asn, Asp, His and Tyr) with queuine. In E. coli this modification occurs via a two-step reaction: (1) TGT-catalyzed base exchange of guanosine-34 with preQ1 (7-aminomethyl-7-deazaguanine) and (2) addition of a cyclopentenediol moiety to the preQ1-34 tRNA. E. coli TGT is normally expressed at very low levels (approximately 1 mg from 500 g cells). The sequence of the queuine operon of E. coli has recently been reported by Reuter et al. (1991). We have cloned the tgt gene into an overexpressing vector in order to provide a more efficient preparation of TGT. A simple, four-step purification scheme yields 78 mg of homogeneous TGT per liter of cell culture (A600 = 5 to 6). Amino-terminal protein sequencing confirms the identity of the recombinant protein and indicates that the initiator methionine is retained in the mature form. Native-PAGE of TGT and SDS-PAGE of cross-linked TGT are most consistent with a hexameric quaternary structure for the enzyme. The cross-linking data also suggests that the enzyme exists as a dimer of trimers of identical 42.5 kDa subunits (total M(r) = 255 kDa. The enzyme is inactivated by cross-linking with the bisimidoester, dimethylsuberimidate. Substrate (tRNA) protects the enzyme against cross-linking and inactivation by dimethylsuberimidate and against inactivation by modification with ethylacetimidate, a monofunctional, imidoester. This indicates that the enzymic residues (presumably lysines) that are involved in cross-linking and the inactivation are in the active site of the enzyme.