RESUMEN
INTRODUCTION: Tattoos are occasionally associated with cutaneous infections. Diagnosis can be challenging as the clinical presentation of such infections may differ from those on plain skin. Herein we report an atypical form of tinea corporis restricted to two recent tattoos during healing, caused by environmental contamination. We reviewed the literature for all cases of fungal infection after tattooing. PATIENTS AND METHODS: A 27-year-old female patient was seen for ring-shaped, erosive, oozing, pruritic and rapidly extensive skin lesions as well as infiltrated papular lesions occurring on tattoos done 6 and 12 days earlier. Fungal analysis revealed Microsporum canis. History-taking indicated that the patient's cat had ringworm and that the patient's sister also had skin lesions consistent with tinea corporis. DISCUSSION: Tinea on tattoos is rarely reported. We found ten additional cases in the literature, as well as five cases of less common fungal infections. These could be explained by the skin break created by the needle during tattooing resulting in an impaired skin barrier, or by accidental self-inoculation (e.g. foot-tattoos). The hypothesis of local immune deficiency induced by tattoo inks strikes us as rather improbable. Unlike usual cases of infections (pyogenic bacteria, mycobacteria, viral hepatitis), fungal infections are not related to a lack of hygiene on the part of the tattooist, but rather to contamination during the healing phase. Their clinical presentation may be atypical, resulting in diagnostic difficulties.
Asunto(s)
Tatuaje , Tiña , Adulto , Animales , Gatos , Femenino , Humanos , Tinta , Microsporum , Piel , Tatuaje/efectos adversos , Tiña/diagnóstico , Tiña/etiología , Tiña/transmisión , ZoonosisRESUMEN
INTRODUCTION: Malakoplakia (MP) is a rare granulomatous disease, usually occurring in immunocompromised patients, linked to Escherichia coli infection. The lesions are usually located in the genitourinary tract, but there is a great variability in the topography and the clinical presentation. CASE REPORT: A 70-year-old diabetic kidney transplant patient under immunosuppressive treatment presented with a voluminous submandibular chronic lesion, involving the skin, associated with a burgeoning lesion of the oral mucosa. Histological examination of biopsies concluded to MP and bacteriological samples were positive for E. coli. Antibiotic treatment allowed for the regression of the lesion before surgical removal. Histological examination of resected material confirmed the diagnosis of invasive MP of the submandibular gland. DISCUSSION: The diagnosis of MP relies on histological examination, showing the presence of von Hansemann's cells and Michaelis- Gutmann bodies. The treatment is based on active antibiotics targeted against intracellular bacteria, possibly associated with surgery. We report the first case of MP involving the submandibular gland.
Asunto(s)
Infecciones por Escherichia coli/patología , Trasplante de Riñón , Malacoplasia/patología , Enfermedades de la Glándula Submandibular/patología , Glándula Submandibular/patología , Anciano , Antibacterianos/uso terapéutico , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/inmunología , Nefropatías Diabéticas/cirugía , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/tratamiento farmacológico , Humanos , Huésped Inmunocomprometido , Malacoplasia/tratamiento farmacológico , Malacoplasia/microbiología , Masculino , Glándula Submandibular/microbiología , Enfermedades de la Glándula Submandibular/tratamiento farmacológico , Enfermedades de la Glándula Submandibular/microbiologíaRESUMEN
Ordered deposition of elongated DNA molecules was achieved by the forced dewetting of a DNA solution droplet over a microstructured substrate. This technique allows trapping, uncoiling, and deposition of DNA fragments without the need of a physicochemical anchoring of the molecule and results in the combing of double stranded DNA from the edge of microwells on a polydimethylsiloxane (PDMS) substrate. The technique involves scanning a droplet of DNA solution caught between a movable blade and a PDMS substrate containing an array of microwells. The deposition and elongation appears when the receding meniscus dewets microwells, the latter acting here as a perturbation in the dewetting line forcing the water film to break locally. Thus, DNA molecules can be deposited in an ordered manner and elongated conformation based solely on a physical phenomenon, allowing uncoiled DNA molecules to be observed in all their length. However, the exact mechanism that governs the deposition of DNA strands is not well understood. This paper is an analysis of the physical phenomenon occurring in the deposition process and is based on observations made with the use of high frame/second rate video microscopy.
RESUMEN
Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK(+) yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8-9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 'early' origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.
Asunto(s)
Bromodesoxiuridina/metabolismo , Fase S , Saccharomyces cerevisiae/metabolismo , Timidina Quinasa/metabolismo , Southern Blotting , Cromatina/genética , Cromatina/metabolismo , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , Replicación del ADN/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Ingeniería Genética , Herpes Simple/enzimología , Herpes Simple/genética , Mutación , Replicón/genética , Saccharomyces cerevisiae/genética , Timidina Quinasa/genéticaRESUMEN
SUN4 is the fourth member of the SUN gene family from S. cerevisiae, whose products display high homology in their 258 amino acid C-terminal domain. SIM1, UTH1, NCA3 (the founding members) are involved in different cellular processes (DNA replication, ageing, mitochondrial biogenesis) and it is shown herein that SUN4 plays a role in the cell septation process. sun4 delta cells are larger than wild-type and begin a new cell cycle before they have separated from their mother cell. This phenotype is more pronounced in sun4Delta cells also deleted for UTH1. FACS analysis shows apparent polyploidy which disappears when the cell cycle is arrested by mating factor or nocodazole, indicating that cell septation is delayed without modification of the doubling time. Elutriated sun4 delta uth1 delta daughter cells are born larger, and therefore enter S phase sooner than their wild-type counterpart. S phase duration, as well as timing of Clb2 degradation, is normal, but cell septation is delayed. Sun4p/Scw3p was recently described as a cell wall protein (Cappellaro et al., 1998) and, consistent with this notion, electron micrographs of sun4 delta cells show defects in the final steps of cell wall septation. Our data suggest that Sun4p and Uth1p might contribute to the regulated process of cell wall morphogenesis and septation.
Asunto(s)
Pared Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/crecimiento & desarrollo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Ciclo Celular , División Celular , Quitinasas/metabolismo , Citometría de Flujo , Proteínas Fúngicas/química , Genes Fúngicos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de la Membrana , Microscopía Electrónica , Proteínas Mitocondriales , Fenotipo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructuraRESUMEN
In all eukaryotes, the initiation of DNA synthesis requires the formation of prereplicative complexes (pre-RCs) on replication origins, followed by their activation by two S-T protein kinases, an S-phase cyclin-dependent kinase (S-CDK) and a homologue of yeast Dbf4-Cdc7 kinase (Dbf4p-dependent kinase [DDK]). Here, we show that yeast DDK activity is cell cycle regulated, though less tightly than that of the S-CDK Clb5-Cdk1, and peaks during S phase in correlation with Dbf4p levels. Dbf4p is short-lived throughout the cell cycle, but its instability is accentuated during G(1) by the anaphase-promoting complex. Downregulating DDK activity is physiologically important, as joint Cdc7p and Dbf4p overexpression is lethal. Because pre-RC formation is a highly ordered process, we asked whether S-CDK and DDK need also to function in a specific order for the firing of origins. We found that both kinases are activated independently, but we show that DDK can perform its function for DNA replication only after S-CDKs have been activated. Cdc45p, a protein needed for initiation, binds tightly to chromatin only after S-CDK activation (L. Zou and B. Stillman, Science 280:593-596, 1998). We show that Cdc45p is phosphorylated by DDK in vitro, suggesting that it might be one of DDK's critical substrates after S-CDK activation. Linking the origin-bound DDK to the tightly regulated S-CDK in a dependent sequence of events may ensure that DNA replication initiates only at the right time and place.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN , Proteínas Fúngicas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Northern Blotting , Western Blotting , Proteína Quinasa CDC2/metabolismo , Proteínas Portadoras/metabolismo , Ciclo Celular , Activación Enzimática , Péptidos y Proteínas de Señalización Intracelular , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Quinasas , Procesamiento Postranscripcional del ARN , Fase S , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
All eukaryotes use similar proteins to licence replication origins but, paradoxically, origin DNA is much less conserved. Specific binding sites for these proteins have now been identified on fission yeast and Drosophila chromosomes, suggesting that the DNA-binding activity of the origin recognition complex has diverged to recruit conserved initiation factors on polymorphic replication origins. Once formed, competent origins are activated by cyclin- and Dbf4-dependent kinases. The latter have been shown to control S phase in several organisms but, in contrast to cyclin-dependent kinases, seem regulated at the level of individual origins. Global and local regulations generate specific patterns of DNA replication that help establish epigenetic chromosome states.
Asunto(s)
Origen de Réplica/genética , Fase S/genética , Animales , Humanos , Modelos Genéticos , Replicón/genéticaRESUMEN
Using a reconstituted DNA replication assay from yeast, we demonstrate that two kinase complexes are essential for the promotion of replication in vitro. An active Clb/Cdc28 kinase complex, or its vertebrate equivalent, is required in trans to stimulate initiation in G(1)-phase nuclei, whereas the Dbf4/Cdc7 kinase complex must be provided by the template nuclei themselves. The regulatory subunit of Cdc7p, Dbf4p, accumulates during late G(1) phase, becomes chromatin associated prior to Clb/Cdc28 activation, and assumes a punctate pattern of localization that is similar to, and dependent on, the origin recognition complex (ORC). The association of Dbf4p with a detergent-insoluble chromatin fraction in G(1)-phase nuclei requires ORC but not Cdc6p or Clb/Cdc28 kinase activity, and correlates with competence for initiation. We propose a model in which Dbf4p targets Cdc7p to the prereplication complex prior to the G(1)/S transition, by a pathway parallel to, but independent of, the Cdc6p-dependent recruitment of MCMs.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , ADN de Hongos/biosíntesis , Proteínas Fúngicas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Origen de Réplica , Proteínas de Saccharomyces cerevisiae , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Fraccionamiento Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Fase G1 , Complejo de Reconocimiento del Origen , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
When yeast cells reach a critical size, they initiate bud formation, spindle pole body duplication, and DNA replication almost simultaneously. All three events depend on activation of Cdc28 protein kinase by the G1 cyclins Cln1, -2, and -3. We show that DNA replication also requires activation of Cdc28 by B-type (Clb) cyclins. A sextuple clb1-6 mutant arrests as multibudded G1 cells that resemble cells lacking the Cdc34 ubiquitin-conjugating enzyme. cdc34 mutants cannot enter S phase because they fail to destroy p40SIC1, which is a potent inhibitor of Clb but not Cln forms of the Cdc28 kinase. In wild-type cells, p40SIC1 protein appears at the end of mitosis and disappears shortly before S phase. Proteolysis of a cyclin-specific inhibitor of Cdc28 is therefore an essential aspect of the G1 to S phase transition.
Asunto(s)
Proteína Quinasa CDC28 de Saccharomyces cerevisiae/fisiología , Ciclo Celular , Ciclina B , Ciclinas/fisiología , Proteínas Fúngicas/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Complejos de Ubiquitina-Proteína Ligasa , Ciclosoma-Complejo Promotor de la Anafase , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Ligasas/fisiología , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína LigasasRESUMEN
We report here the sequence of RPK1 (for Regulatory cell Proliferation Kinase), a new Saccharomyces cerevisiae gene coding for a protein with sequence similarities to serine/threonine protein kinases. The protein sequence of 764 amino acids includes an amino-terminal domain (residues 1-410), which may be involved in regulation of the kinase domain (residues 411-764). The catalytic domain of Rpk1 is not closely related to other known yeast protein kinases but exhibits strong homology to a newly discovered group of mammalian kinases (PYT, TTK, esk) with serine/threonine/tyrosine kinase activity. Null alleles of RPK1 are lethal and thus this gene belongs to the small group of yeast protein kinase genes that are essential for cell growth. In addition, eliminating the expression of RPK1 gives rise to the accumulation of non-viable cells with less than a 1 N DNA content suggesting that cells proceed into mitosis without completion of DNA synthesis. Therefore, the Rpk1 kinase may function in a checkpoint control which couples DNA replication to mitosis. The level of the RPK1 transcript is extremely low and constant throughout the mitotic cycle. However it is regulated during cellular differentiation, being decreased in alpha-factor-treated a cells and increased late in meiosis in a/alpha diploids. Taken together, our results suggest that Rpk1 is involved in a pathway that coordinates cell proliferation and differentiation.
Asunto(s)
División Celular/genética , Genes Fúngicos/genética , Genes Letales/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Diferenciación Celular/genética , ADN de Hongos/biosíntesis , Regulación Fúngica de la Expresión Génica , Factor de Apareamiento , Meiosis/genética , Mitosis/genética , Datos de Secuencia Molecular , Péptidos/genética , Feromonas/farmacología , ARN Mensajero/biosíntesis , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The evolution of the aminoacyl-tRNA synthetases is intriguing in light of their elaborate relationship with tRNAs and their significance in the decoding process. Based on sequence motifs and structure determination, these enzymes have been assigned to two classes. The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS), a class I enzyme, complexed to tRNA(Gln) and ATP has been described. It is shown here that a 'minimal' GlnRS, i.e. a GlnRS from which domains interacting with the acceptor-end and the anticodon of the tRNA have been deleted, has enzymatic activity and can charge a tRNA(Tyr)-derived amber suppressor (supF) with glutamine. The catalytic core of GlnRS, which is structurally conserved in other class I synthetases, is therefore sufficient for the aminoacylation of tRNA substrates. Some of these truncated enzymes have lost their ability to discriminate against non-cognate tRNAs, implying a more specific role of the acceptor-end-binding domain in the recognition of tRNAs. Our results indicate that the catalytic and substrate recognition properties are carried by distinct domains of GlnRS, and support the notion that class I aminoacyl-tRNA synthetases evolved from a common ancestor, jointly with tRNAs and the genetic code, by the addition of non-catalytic domains conferring new recognition specificities.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Aminoacilación de ARN de Transferencia , Proteínas Bacterianas/química , Secuencia de Bases , Sitios de Unión , Evolución Biológica , Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , ARN Bacteriano/genética , ARN de Transferencia de Glutamina/metabolismo , ARN de Transferencia de Serina/metabolismo , Relación Estructura-ActividadRESUMEN
The site I 22 kDa auxin-binding proteins from maize are encoded by a small gene family comprising at least five members. Here the cloning and molecular analysis of the Zm-ERabp1, Zm-ERabp4, and Zm-ERabp5 genes is presented. All three encode 22-23 kDa proteins displaying a transit peptide, a C-terminal KDEL sequence, as well as glycosylation and auxin-binding sites. The Zm-ERabp4 and Zm-ERabp5 genes are very similar. The Zm-ERabp1 gene encodes a related protein, but its promoter, leader and signal peptide are very different. Northern analysis using gene-specific oligonucleotide probes indicates that Zm-ERabp4 is expressed in leaves and coleoptiles but weakly in roots, whereas Zm-ERabp5 expression is barely detectable in these tissues. RNA-PCR indicated that all three genes are none the less expressed in many tissues. Primer-extension analysis revealed an unusually long (320 bases) Zm-ERabp1 leader containing an 80 codon ORF which, if expressed, would encode a positively charged protein with some similarity to transcription factors. In a transient promoter-reporter gene expression system using maize leaf protoplasts the Zm-ERabp1 promoter is more active than the Zm-ERabp4 and Zm-ERabp5 promoters. Promoter deletion analysis of Zm-ERabp1 has identified a negative regulatory sequence in a region from -364 bp and -130 bp, deletion of which results in about twofold higher expression. This region contains both enhancer- and G-box-related sequences. Deletion of -126 bp to +64 bp, which contains the TATA box and transcription start, results in a large decrease in expression.
Asunto(s)
Genes de Plantas , Reguladores del Crecimiento de las Plantas , Receptores de Superficie Celular/genética , Zea mays/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Expresión Génica , Genes Reguladores , Genes Reporteros , Ácidos Indolacéticos/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , ARN/genética , Receptores de Superficie Celular/metabolismo , Mapeo Restrictivo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Distribución Tisular , Zea mays/metabolismoRESUMEN
The functions of the Cdc28 protein kinase in DNA replication and mitosis in Saccharomyces cerevisiae are thought to be determined by the type of cyclin subunit with which it is associated. G1-specific cyclins encoded by CLN1, CLN2, and CLN3 are required for entry into the cell cycle (Start) and thereby for S phase, whereas G2-specific B-type cyclins encoded by CLB1, CLB2, CLB3, and CLB4 are required for mitosis. We describe a new family of B-type cyclin genes, CLB5 and CLB6, whose transcripts appear in late G1 along with those of CLN1, CLN2, and many genes required for DNA replication. Deletion of CLB6 has little or no effect, but deletion of CLB5 greatly extends S phase, and deleting both genes prevents the timely initiation of DNA replication. Transcription of CLB5 and CLB6 is normally dependent on Cln activity, but ectopic CLB5 expression allows cells to proliferate in the absence of Cln cyclins. Thus, the kinase activity associated with Clb5/6 and not with Cln cyclins may be responsible for S-phase entry. Clb5 also has a function, along with Clb3 and Clb4, in the formation of mitotic spindles. Our observation that CLB5 is involved in the initiation of both S phase and mitosis suggests that a single primordial B-type cyclin might have been sufficient for regulating the cell cycle of the common ancestor of many, if not all, eukaryotes.
Asunto(s)
Ciclinas/genética , Replicación del ADN/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Fase S/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia de Consenso , Secuencia Conservada , Ciclinas/química , Ciclinas/fisiología , Eliminación de Gen , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , ARN Mensajero/análisis , Huso Acromático , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Unlike early embryonic cleavage divisions in certain animals, cell-cycle progression in yeast and probably also in all metazoan somatic cells requires the periodic transcriptional activation of certain key genes. Thus far, the only clear examples are genes that encode a class of unstable 'cyclin' proteins, which bind and activate the cdc2/Cdc28 protein kinase: the G1-specific cyclins encoded by CLN1 and CLN2, a B-type cyclin implicated in DNA replication encoded by CLB5; and four B-type cyclins involved in mitosis encoded by CLB1, 2, 3, 4. CLN1, CLN2, and CLB5 are transcribed in late G1, as cells undergo Start. A transcription factor composed of Swi4 and Swi6 proteins (called SBF) activates CLN1 and CLN2 transcription via a positive feedback loop in which Cln proteins activate their own transcription. A different but related transcription factor called MBF seems responsible for the late G1-specific transcription of most DNA replication genes including CLB5. We have purified MBF and shown that it contains Swi6 and a 110-120 kDa protein distinct from Swi4 (p120) that contacts DNA. Thus, we propose that SBF and MBF share a common regulatory subunit (Swi6) but recognize their promoter elements via distinct DNA binding subunits.
Asunto(s)
Ciclo Celular , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclinas/metabolismo , Replicación del ADN , Retroalimentación , Datos de Secuencia Molecular , Peso Molecular , Sondas de Oligonucleótidos , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genéticaRESUMEN
The recognition of the acceptor stem of tRNA(Gln) is an important element ensuring the accuracy of aminoacylation by Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18). On the basis of known mutations and the crystal structure of the tRNA(Gln).GlnRS complex, we mutagenized at saturation two motifs in the acceptor end binding domain of GlnRS. Mutants with lowered tRNA specificity were then selected in vivo by suppression of a glutamine-specific amber mutation (lacZ1000) with an amber suppressor tRNA derived from tRNA(1Ser). The mischarging GlnRS mutants obtained in this way retain the ability to charge tRNA(Gln), but in addition, they misacylate a number of noncognate amber suppressor tRNAs. The critical residues responsible for specificity are Arg-130 and Glu-131, located in a part of GlnRS that binds the acceptor stem of tRNA(Gln). On the basis of the spectrum of tRNAs capable of being misacylated by such mutants we propose that, in addition to taking part in productive interactions, the acceptor end binding domain contributes to recognition specificity by rejecting noncognate tRNAs through negative interactions. Analysis of the catalytic properties of one of the mischarging enzymes, GlnRS100 (Arg-130-->Pro, Glu-131-->Asp), indicates that, while the kinetic parameters of the mutant enzyme are not dramatically changed, it binds noncognate tRNA(Glu) more stably than the wild-type enzyme does (Kd is 1/8 that of the wild type). Thus, the stability of the noncognate complex may be the basis for mischarging in vivo.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , ARN de Transferencia de Glutamina/metabolismo , Aminoacilación de ARN de Transferencia , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , ARN Bacteriano/metabolismo , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
The specific recognition by Escherichia coli glutaminyl-tRNA synthetase (GlnRS) of tRNA(Gln) is mediated by extensive protein:RNA contacts and changes in the conformation of tRNA(Gln) when complexed with GlnRS. In vivo accuracy of aminoacylation depends on two factors: competition between synthetases, and the context and recognition of identity elements in the tRNA. The structure of the tRNA(Gln):GlnRS complex supports studies from amber and opal suppressor tRNAs, complemented by in vitro aminoacylation of the mutated tRNA transcripts, that the glutamine identity elements are located in the anticodon and acceptor stem of tRNA(Gln). Recognition of individual functional groups in tRNA, for example the 2-amino group of guanosine, is also evident from the result with inosine-substituted tRNAs. Communication between anticodon and acceptor stem recognition is indicated by mutants in GlnRS isolated by genetic selection with opal suppressor tRNAs which are altered in interactions with the inside of the L-shaped tRNA. We have also used genetic selection to obtain mutants of GlnRS altered in acceptor stem recognition with relaxed specificity for amber suppressor tRNAs, and a more extensive mutational analysis shows the importance of the acceptor binding domain to accurate recognition of tRNA.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Escherichia coli/enzimología , ARN de Transferencia de Glutamina/metabolismo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Anticodón/química , Anticodón/metabolismo , Secuencia de Bases , Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Conformación de Ácido Nucleico , Conformación Proteica , ARN de Transferencia de Glutamina/química , ARN de Transferencia de Glutamina/genética , Especificidad por SustratoRESUMEN
A variety of genetic, biochemical and structural studies have been used to determine factors ensuring the accuracy of recognition by aminoacyl-tRNA synthetases for tRNA. The identity elements of Escherichia coli tRNA(Gln) are located mainly in the anticodon and acceptor stem, and ensure the accurate recognition of the tRNA by glutaminyl-tRNA synthetase. We summarize a number of experimental techniques to define the accuracy of aminoacylation in vivo and in vitro.