RESUMEN
The process of blood formation, haematopoiesis, depends upon a small number of haematopoietic stem cells (HSCs) that reside in the bone marrow. Differentiation of HSCs is characterised by decreased expression of genes associated with self-renewal accompanied by a stepwise activation of genes promoting differentiation. Lineage branching is further directed by groups of cooperating and counteracting genes forming complex networks of lineage-specific transcription factors. Imbalances in such networks can result in blockage of differentiation, lineage reprogramming and malignant transformation. CCAAT/enhancer-binding protein-α (C/EBPα) was originally identified 30 years ago as a transcription factor that binds both promoter and enhancer regions. Most of the early work focused on the role of C/EBPα in regulating transcriptional processes as well as on its functions in key differentiation processes during liver, adipogenic and haematopoietic development. Specifically, C/EBPα was shown to control differentiation by its ability to coordinate transcriptional output with cell cycle progression. Later, its role as an important tumour suppressor, mainly in acute myeloid leukaemia (AML), was recognised and has been the focus of intense studies by a number of investigators. More recent work has revisited the role of C/EBPα in normal haematopoiesis, especially its function in HSCs, and also started to provide more mechanistic insights into its role in normal and malignant haematopoiesis. In particular, the differential actions of C/EBPα isoforms, as well as its importance in chromatin remodelling and cellular reprogramming, are beginning to be elucidated. Finally, recent work has also shed light on the dichotomous function of C/EBPα in AML by demonstrating its ability to act as both a tumour suppressor and promoter. In the present review, we will summarise the current knowledge on the functions of C/EBPα during normal and malignant haematopoiesis with special emphasis on the recent work.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Neoplasias Hematológicas/fisiopatología , Hematopoyesis/fisiología , Animales , HumanosAsunto(s)
Aberraciones Cromosómicas , Células Madre Hematopoyéticas/patología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Monocitos/patología , Células Mieloides/patología , Estudios de Casos y Controles , Cromosomas Humanos Par 15/genética , Cromosomas Humanos Par 17/genética , Citometría de Flujo , Estudios de Seguimiento , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunofenotipificación , Hibridación Fluorescente in Situ , Monocitos/metabolismo , Células Mieloides/metabolismo , PronósticoRESUMEN
Members of the TALE (three-amino-acid loop extension) family of atypical homeodomain-containing transcription factors are important downstream effectors of oncogenic fusion proteins involving the mixed lineage leukemia (MLL) gene. A well-characterized member of this protein family is MEIS1, which orchestrates a transcriptional program required for the maintenance of MLL-rearranged acute myeloid leukemia (AML). TGIF1/TGIF2 are relatively uncharacterized TALE transcription factors, which, in contrast to the remaining family, have been shown to act as transcriptional repressors. Given the general importance of this family in malignant hematopoiesis, we therefore tested the potential function of TGIF1 in the maintenance of MLL-rearranged AML. Gene expression analysis of MLL-rearranged patient blasts demonstrated reduced TGIF1 levels, and, in accordance, we find that forced expression of TGIF1 in MLL-AF9-transformed cells promoted differentiation and cell cycle exit in vitro, and delayed leukemic onset in vivo. Mechanistically, we show that TGIF1 interferes with a MEIS1-dependent transcriptional program by associating with MEIS1-bound regions in a competitive manner and that the MEIS1:TGIF1 ratio influence the clinical outcome. Collectively, these findings demonstrate that TALE family members can act both positively and negatively on transcriptional programs responsible for leukemic maintenance and provide novel insights into the regulatory gene expression circuitries in MLL-rearranged AML.
Asunto(s)
Regulación Leucémica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , Proteínas de Homeodominio/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Represoras/metabolismo , Animales , Células de la Médula Ósea/citología , Ciclo Celular , Diferenciación Celular , Inmunoprecipitación de Cromatina , Citometría de Flujo , Perfilación de la Expresión Génica , Genes Homeobox , Humanos , Ratones , Ratones Endogámicos C57BL , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Factor de Crecimiento Transformador beta1/metabolismo , Resultado del TratamientoRESUMEN
Reciprocal chromosomal translocations are observed in one-third of acute myeloid leukemia (AML) cases. Targeting and understanding the effects of the resulting aberrant oncogenic fusion proteins may help developing drugs against specific leukemic subtypes, as demonstrated earlier by the use of ATRA in acute promyelocytic leukemia. Hematopoietic stem/progenitor (HSPCs) cells transduced with oncogenic fusion genes are regarded as promising in vitromodels of their corresponding AML subtypes. Here, we critically assessed the potential of such in vitro models using an integrative bioinformatics approach. Surprisingly, we found that the gene-expression profiles of CD34+ human HSPCs transformed with the potent oncogenic fusion proteins AML-ETO or MLL-AF9, only weakly resembled those derived from primary AML samples. Hence, our work raises concerns as to the relevance of the use of in vitro transduced cells to study the impact of transcriptional deregulation in human AML.
RESUMEN
Chromosomal translocations of transcription factors generating fusion proteins with aberrant transcriptional activity are common in acute leukemia. In acute promyelocytic leukemia (APL), the promyelocytic leukemia-retinoic-acid receptor alpha (PML-RARA) fusion protein, which emerges as a consequence of the t(15;17) translocation, acts as a transcriptional repressor that blocks neutrophil differentiation at the promyelocyte (PM) stage. In this study, we used publicly available microarray data sets and identified signatures of genes dysregulated in APL by comparison of gene expression profiles of APL cells and normal PMs representing the same stage of differentiation. We next subjected our identified APL signatures of dysregulated genes to a series of computational analyses leading to (i) the finding that APL cells show stem cell properties with respect to gene expression and transcriptional regulation, and (ii) the identification of candidate drugs and drug targets for therapeutic interventions. Significantly, our study provides a conceptual framework that can be applied to any subtype of AML and cancer in general to uncover novel information from published microarray data sets at low cost. In a broader perspective, our study provides strong evidence that genomic strategies might be used in a clinical setting to prospectively identify candidate drugs that subsequently are validated in vitro to define the most effective drug combination for individual cancer patients on a rational basis.
Asunto(s)
Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Leucemia Promielocítica Aguda/genética , Tretinoina/farmacología , Células Cultivadas , Perfilación de la Expresión Génica , Células Precursoras de Granulocitos/efectos de los fármacos , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The C/EBPalpha transcription factor is required for differentiation of adipocytes and neutrophil granulocytes, and controls cellular proliferation in vivo. To address the molecular mechanisms of C/EBPalpha action, we have identified C/EBPalpha mutants defective in repression of E2F-dependent transcription and found them to be impaired in their ability to suppress cellular proliferation, and to induce adipocyte differentiation in vitro. Using targeted mutagenesis of the mouse germline, we show that E2F repression-deficient C/EBPalpha alleles failed to support adipocyte and granulocyte differentiation in vivo. These results indicate that E2F repression by C/EBPalpha is critical for its ability to induce terminal differentiation, and thus provide genetic evidence that direct cell cycle control by a mammalian lineage-instructive transcription factor couples cellular growth arrest and differentiation.
Asunto(s)
Adipocitos/citología , Proteína alfa Potenciadora de Unión a CCAAT/química , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Granulocitos/citología , Factores de Transcripción/química , Células 3T3 , Alelos , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting , Diferenciación Celular , División Celular , Factores de Transcripción E2F , Femenino , Citometría de Flujo , Genes Reporteros , Glutatión Transferasa/metabolismo , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ovario/metabolismo , Unión Proteica , Ratas , Homología de Secuencia de Aminoácido , Distribución Tisular , Transcripción GenéticaRESUMEN
The binding site of puromycin was probed chemically in the peptidyl-transferase center of ribosomes from Escherichia coli and of puromycin-hypersensitive ribosomes from the archaeon Haloferax gibbonsii. Several nucleotides of the 23S rRNAs showed altered chemical reactivities in the presence of puromycin. They include A2439, G2505, and G2553 for E. coli, and G2058, A2503, G2505, and G2553 for Hf. gibbonsii (using the E. coli numbering system). Reproducible enhanced reactivities were also observed at A508 and A1579 within domains I and III, respectively, of E. coli 23S rRNA. In further experiments, puromycin was shown to produce a major reduction in the UV-induced crosslinking of deacylated-(2N3A76)tRNA to U2506 within the P' site of E. coli ribosomes. Moreover, it strongly stimulated the putative UV-induced crosslink between a streptogramin B drug and m2A2503/psi2504 at an adjacent site in E. coli 23S rRNA. These data strongly support the concept that puromycin, along with other peptidyl-transferase antibiotics, in particular the streptogramin B drugs, bind to an RNA structural motif that contains several conserved and accessible base moieties of the peptidyl transferase loop region. This streptogramin motif is also likely to provide binding sites for the 3' termini of the acceptor and donor tRNAs. In contrast, the effects at A508 and A1579, which are located at the exit site of the peptide channel, are likely to be caused by a structural effect transmitted along the peptide channel.
Asunto(s)
Peptidil Transferasas/metabolismo , Puromicina/metabolismo , ARN Ribosómico/metabolismo , Secuencia de Bases , Sitios de Unión , Escherichia coli/genética , Escherichia coli/metabolismo , Haloferax/genética , Haloferax/metabolismo , Datos de Secuencia Molecular , Peptidil Transferasas/química , Puromicina/química , ARN de Archaea/química , ARN de Archaea/genética , ARN de Archaea/metabolismo , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Ribosómico/química , ARN Ribosómico/genética , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Especificidad por SustratoRESUMEN
A range of antibiotic inhibitors that act within the peptidyl transferase center of the ribosome were examined for their capacity to perturb the relative positioning of the 3' end of P/P'-site-bound tRNA and the Escherichia coli ribosome. The 3'-terminal adenosines of deacylated tRNA and N-Ac-Phe-tRNA were derivatized at the 2 position with an azido group and the tRNAs were cross-linked to the ribosome on irradiation with ultraviolet light at 365 nm. The cross-links were localized on the rRNA within extended versions of three previously characterized 23S rRNA fragments F1', F2', and F4' at nucleotides C2601/A2602, U2584/U2585 (F1'), U2506 (F2'), and A2062/C2063 (F4'). Each of these nucleotides lies within the peptidyl transferase loop region of the 23S rRNA. Cross-links were also formed with ribosomal proteins L27 (strong) and L33 (weak), as shown earlier. The antibiotics sparsomycin, chloramphenicol, the streptogramins pristinamycin IA and IIA, gougerotin, lincomycin, and spiramycin were tested for their capacity to alter the identities or yields of each of the cross-links. Although no new cross-links were detected, each of the drugs produced major changes in cross-linking yields, mainly decreases, at one or more rRNA sites but, with the exception of chloramphenicol, did not affect cross-linking to the ribosomal proteins. Moreover, the effects were closely similar for both deacylated and N-Ac-Phe-tRNAs, indicating that the drugs selectively perturb the 3' terminus of the tRNA. The strongest decreases in the rRNA cross-links were observed with pristinamycin IIA and chloramphenicol, which correlates with their both producing complex chemical footprints on 23S rRNA within E. coli ribosomes. Furthermore, gougerotin and pristinamycin IA strongly increased the yields of fragments F2' (U2506) and F4' (U2062/C2063), respectively. The results obtained with an RNAse H approach correlate well with primer extension data implying that cross-linking occurs primarily to the bases. Both sets of data are also consistent with the results of earlier rRNA footprinting experiments on antibiotic-ribosome complexes. It is concluded that the antibiotics perturb the relative positioning of the 3' end of the P/P'-site-bound tRNA and the peptidyl transferase loop region of 23S rRNA.
Asunto(s)
Adenosina/metabolismo , Antibacterianos/farmacología , Peptidil Transferasas/farmacología , ARN Ribosómico 23S/efectos de los fármacos , ARN de Transferencia de Fenilalanina/efectos de los fármacos , Ribosomas/efectos de los fármacos , Antibióticos Antineoplásicos/farmacología , Autorradiografía , Cloranfenicol/farmacología , Reactivos de Enlaces Cruzados/farmacología , Escherichia coli/enzimología , Modelos Genéticos , Inhibidores de la Síntesis de la Proteína/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Rayos Ultravioleta , Virginiamicina/farmacologíaRESUMEN
The antitumor antibiotic sparsomycin is a universal and potent inhibitor of peptide bond formation and selectively acts on several human tumors. It binds to the ribosome strongly, at an unknown site, in the presence of an N-blocked donor tRNA substrate, which it stabilizes on the ribosome. Its site of action was investigated by inducing a crosslink between sparsomycin and bacterial, archaeal, and eukaryotic ribosomes complexed with P-site-bound tRNA, on irradiating with low energy ultraviolet light (at 365 nm). The crosslink was localized exclusively to the universally conserved nucleotide A2602 within the peptidyl transferase loop region of 23S-like rRNA by using a combination of a primer extension approach, RNase H fragment analysis, and crosslinking with radioactive [(125)I]phenol-alanine-sparsomycin. Crosslinking of several sparsomycin derivatives, modified near the sulfoxy group, implicated the modified uracil residue in the rRNA crosslink. The yield of the antibiotic crosslink was weak in the presence of deacylated tRNA and strong in the presence of an N-blocked P-site-bound tRNA, which, as was shown earlier, increases the accessibility of A2602 on the ribosome. We infer that both A2602 and its induced conformational switch are critically important both for the peptidyl transfer reaction and for antibiotic inhibition. This supposition is reinforced by the observation that other antibiotics that can prevent peptide bond formation in vitro inhibit, to different degrees, formation of the crosslink.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Escherichia coli/metabolismo , Peptidil Transferasas/metabolismo , ARN Ribosómico 23S/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Esparsomicina/análogos & derivados , Esparsomicina/metabolismo , Antibióticos Antineoplásicos/farmacología , Bacillus megaterium/metabolismo , Secuencia de Bases , Reactivos de Enlaces Cruzados/farmacología , Halobacterium salinarum/metabolismo , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Peptidil Transferasas/química , ARN Bacteriano/metabolismo , ARN de Hongos/metabolismo , ARN Ribosómico 23S/química , ARN de Transferencia/química , Ribosomas/efectos de los fármacos , Ribosomas/ultraestructura , Saccharomyces cerevisiae/metabolismo , Esparsomicina/farmacologíaAsunto(s)
Antibacterianos/metabolismo , Guanosina Trifosfato/metabolismo , Conformación de Ácido Nucleico , Conformación Proteica , ARN Ribosómico 23S/química , Proteínas Ribosómicas/química , Sitios de Unión , Hidrólisis , Factores de Elongación de Péptidos/metabolismo , ARN Ribosómico 23S/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas , Thermotoga maritima/genética , Thermotoga maritima/metabolismoRESUMEN
The naturally occurring streptogramin B antibiotic, pristinamycin IA, which inhibits peptide elongation, can produce two modifications in 23S rRNA when bound to the Escherichia coli 70S ribosome and irradiated at 365 nm. Both drug-induced effects map to highly conserved nucleotides within the functionally important peptidyl transferase loop of 23S rRNA at positions m2A2503/psi2504 and G2061/A2062. The modification yields are influenced strongly, and differentially, by P-site-bound tRNA and strongly by some of the peptidyl transferase antibiotics tested, with chloramphenicol producing a shift in the latter modification to A2062/C2063. Pristinamycin IA can also produce a modification on binding to deproteinized, mature 23S rRNA, at position U2500/C2501. The same modification occurs on an approximately 37-nt fragment, encompassing positions approximately 2496-2532 of the peptidyl transferase loop that was excised from the mature rRNA using RNAse H. In contrast, no antibiotic-induced effects were observed on in vitro T7 transcripts of full-length 23S rRNA, domain V, or on a fragment extending from positions approximately 2496-2566, which indicates that one or more posttranscriptional modifications within the sequence Cm-C-U-C-G-m2A-psi-G2505 are important for pristinamycin IA binding and/or the antibiotic-dependent modification of 23S rRNA.
Asunto(s)
Antibacterianos/metabolismo , Escherichia coli/genética , Peptidil Transferasas/genética , ARN Ribosómico 23S/genética , Virginiamicina/metabolismo , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Estructura Molecular , Peptidil Transferasas/efectos de la radiación , Procesamiento Postranscripcional del ARN/efectos de la radiación , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Ribonucleasa H/metabolismo , Rayos UltravioletaRESUMEN
Micrococcin-resistant mutants of Bacillus megaterium that carry mutations affecting ribosomal protein L11 have been characterised. The mutants fall into two groups. "L11-minus" strains containing an L11 gene with deletions, insertions or nonsense mutations which grow 2.5-fold slower than the wild-type strain, whereas other mutants carrying single-site substitutions within an 11 amino acid residue segment of the N-terminal domain of L11 grow normally. Protein L11 binds to 23 S rRNA within the ribosomal GTPase centre which regulates GTP hydrolysis on ribosomal factors. Micrococcin binding within the rRNA component of this centre was probed on wild-type and mutant ribosomes, in vivo, using dimethyl sulphate where it generated an rRNA footprint indistinguishable from that produced in vitro, even after the cell growth had been arrested by treatment with either kirromycin or fusidic acid. No drug-rRNA binding was detected in vivo for the L11-minus mutants, while reduced binding (approximately 30-fold) was observed for two single-site mutants P23L and P26L. For the latter, the reduced drug affinity alone did not account for the resistance-phenotype because rapid cell growth occurred even at drug concentrations that would saturate the ribosomes. Micrococcin was also bound to complexes containing an rRNA fragment and wild-type or mutant L11, expressed as fusion proteins, and they were probed with proteinases. The drug produced strong protection effects on the wild-type protein and weak effects on the P23L and P26L mutant proteins. We infer that inhibition of cell growth by micrococcin, as for thiostrepton, results from the imposition of a conformational constraint on protein L11 which, in turn, perturbs the function(s) of the ribosomal factor-guanosine nucleotide complexes.
Asunto(s)
Antibacterianos/farmacología , Bacillus megaterium/genética , GTP Fosfohidrolasas , Péptidos , Proteínas Ribosómicas/efectos de los fármacos , Ribosomas/efectos de los fármacos , Secuencia de Aminoácidos , Antibacterianos/metabolismo , Bacteriocinas , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Farmacorresistencia Microbiana/genética , Ácido Fusídico/farmacología , Datos de Secuencia Molecular , Mutación , Inhibidores de la Síntesis de la Proteína/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Piridonas/farmacología , ARN Ribosómico 23S/metabolismo , Proteínas de Unión al ARN/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Análisis de Secuencia de ADN , Tioestreptona/farmacologíaRESUMEN
Streptogramin antibiotics contain two active A and B components that inhibit peptide elongation synergistically. Mutants resistant to the A component (virginiamycin M1 and pristinamycin IIA) were selected for the archaeon Halobacterium halobium. The mutations mapped to the universally conserved nucleotides A2059 and A2503 within the peptidyl transferase loop of 23 S rRNA (Escherichia coli numbering). When bound to wild-type and mutant haloarchaeal ribosomes, the A and B components (pristinamycins IIA and IA, respectively) produced partially overlapping rRNA footprints, involving six to eight nucleotides in the peptidyl transferase loop of 23 S rRNA, including the two mutated nucleotides. An rRNA footprinting study, performed both in vivo and in vitro, on the A and B components complexed to Bacillus megaterium ribosomes, indicated that similar drug-induced effects occur on free ribosomes and within the bacterial cells. It is inferred that position 2058 and the sites of mutation, A2059 and A2503, are involved in the synergistic inhibition by the two antibiotics. A structural model is presented which links A2059 and A2503 and provides a structural rationale for the rRNA footprints.
Asunto(s)
Halobacterium salinarum/efectos de los fármacos , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , ARN Ribosómico 23S/química , Virginiamicina/farmacología , Bacillus megaterium/efectos de los fármacos , Bacillus megaterium/ultraestructura , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cloranfenicol/farmacología , Farmacorresistencia Microbiana , Sinergismo Farmacológico , Halobacterium salinarum/genética , Halobacterium salinarum/crecimiento & desarrollo , Sustancias Macromoleculares , Modelos Biológicos , Conformación de Ácido Nucleico , Peptidil Transferasas/metabolismo , Mutación Puntual , ARN Ribosómico 23S/efectos de los fármacos , Ribosomas/efectos de los fármacosRESUMEN
The six major structural domains of 23 S rRNA from Escherichia coli, and all combinations thereof, were synthesized as separate T7 transcripts and reconstituted with total 50 S subunit proteins. Analysis by one and two-dimensional gel electrophoresis demonstrated the presence of at least one primary binding protein associated with each RNA domain and additional proteins assembled to domains I, II, V and VI. For all the combinations of two to five domains, enhanced assembly yields and/or new proteins were observed primarily to those transcripts containing either domains I+II or domains V+VI. This indicates that there are two major protein assembly centres located at the ends of the 23 S rRNA, which is consistent with an earlier view that in vitro protein assembly nucleates around proteins L24 and L3. Although similar protein assembly patterns were observed over a range of temperature and magnesium concentrations, protein L2 assembled strongly with domains II and IV at 4-8 mM Mg2+ (the first step of the two-step reconstitution procedure) and with domain IV alone at higher Mg2+ concentrations (the second step). It is proposed that this change in protein-RNA binding provides a basis for the two-step reconstitution in vitro. A chemical footprinting approach was employed on the reconstituted protein-domain complexes to localize a putative L4 binding region within domain I to a region that is partially co-structural with the site on the L4-mRNA where L4 binds and inhibits its own translation. A similar approach was used to map the putative binding regions on domain V of protein L9 and the 5 S RNA-L5-L18 complex.
Asunto(s)
ARN Bacteriano/metabolismo , ARN Ribosómico 23S/metabolismo , ARN Ribosómico 5S/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Secuencia de Bases , Sitios de Unión , Escherichia coli , Iones , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Proteína Ribosomal L3 , TemperaturaRESUMEN
A newly identified class of highly thiostrepton-resistant mutants of the archaeon Halobacterium halobium carry a missense mutation at codon 18 within the gene encoding ribosomal protein L11. In the mutant proteins, a proline, conserved in archaea and bacteria, is converted to either serine or threonine. The mutations do not impair either the assembly of the mutant L11 into 70 S ribosomes in vivo or the binding of thiostrepton to ribosomes in vitro. Moreover, the corresponding mutations at proline 22, in a fusion protein of L11 from Escherichia coli with glutathione-S-transferase, did not reduce the binding affinities of the mutated L11 fusion proteins for rRNA of of thiostrepton for the mutant L11-rRNA complexes at rRNA concentrations lower than those prevailing in vivo. Probing the structure of the fusion protein of wild-type L11, from E. coli, using a recently developed protein footprinting technique, demonstrated that a general tightening of the C-terminal domain occurred on rRNA binding, while thiostrepton produced a footprint centred on tyrosine 62 at the junction of the N and C-terminal domains of protein L11 complexed to rRNA. The intensity of this protein footprint was strongly reduced for the mutant L11-rRNA complexes. These results indicate that although, as shown earlier, thiostrepton binds primarily to 23 S rRNA, the drug probably inhibits peptide elongation by impeding a conformational change within protein L11 that is important for the function of the ribosomal GTPase centre. This putative inhibitory mechanism of thiostrepton is critically dependent on proline 18/22. Moreover, the absence of this proline from eukaryotic protein L11 sequences would account for the high thiostrepton resistance of eukaryotic ribosomes.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Halobacterium salinarum/efectos de los fármacos , Proteínas Ribosómicas/genética , Tioestreptona/farmacología , Secuencia de Aminoácidos , Antibacterianos/farmacología , Huella de ADN , Farmacorresistencia Microbiana/genética , Halobacterium salinarum/genética , Datos de Secuencia Molecular , Mutación , ARN/metabolismo , ARN Ribosómico 23S/química , ARN Ribosómico 23S/efectos de los fármacos , ARN Ribosómico 23S/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Ribosómicas/efectos de los fármacos , Proteínas Ribosómicas/metabolismo , Ribosomas/efectos de los fármacos , Tioestreptona/metabolismoRESUMEN
Determining how antibiotics inhibit ribosomal activity requires a detailed understanding of the interactions and relative movement of tRNA, mRNA and the ribosome. Recent models for the formation of hybrid tRNA binding sites during the elongation cycle have provided a basis for re-evaluating earlier experimental data and, especially, those relevant to substrate movements through the peptidyl transferase centre. With the exception of deacylated tRNA, which binds at the E-site, ribosomal interactions of the 3'-ends of the tRNA substrates generate only a small part of the total free energy of tRNA-ribosome binding. Nevertheless, these relatively weak interactions determine the unidirectional movement of tRNAs through the ribosome and, moreover, they appear to be particularly susceptible to perturbation by antibiotics. Here we summarise current ideas relating particularly to the movement of the 3'-ends of tRNA through the ribosome and consider possible inhibitory mechanisms of the peptidyl transferase antibiotics.
Asunto(s)
Antibacterianos/farmacología , Peptidil Transferasas/metabolismo , ARN Bacteriano/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Antibacterianos/metabolismo , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Péptidos/metabolismo , Aminoacil-ARN de Transferencia/metabolismoRESUMEN
An RNA region associated with the donor substrate site, located at the base of the peptidyl transferase loop of 23 S rRNA, was subjected to a comprehensive single-site mutational study. Growth phenotypes of Escherichia coli cells were characterized on induction of synthesis of the mutated rRNAs and the mutated ribosomes were tested, selectively, for their capacity to generate peptide bonds under the conditions of the "fragment" assay. Most of the mutants exhibited dominant or recessive lethal growth phenotypes and, in general, defective growth correlated with low activities in peptide bond formation, although exceptions were observed with normal growth and low activities, and vice versa. All these phenotypes are consistent with defects occurring in the structure of the ribosomal donor site and/or the capacity of the donor substrate to enter or leave this site. A compensating base change approach was employed to test for Watson-Crick base-pairing interactions between the -CCA end of the P-site bound tRNA(Phe) and this region of the peptidyl-transferase loop. Single nucleotide substitutions were introduced into the -CCA end of tRNA(Phe) and the ability of the 3'-terminal pentanucleotide fragments to act as donor substrates was examined for ribosomes carrying the different mutated 23 S rRNAs. No evidence was found for the occurrence of Watson-Crick base-pairing interactions. However, the data are consistent with the formation of a Hoogsteen pair between the 3'-terminal adenosine base of the donor substrate and U2585 of the 23 S rRNA.
Asunto(s)
Extensión de la Cadena Peptídica de Translación/genética , Peptidil Transferasas/metabolismo , Mutación Puntual , ARN Ribosómico 23S/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , ARN Ribosómico 23S/genética , Ribonucleósidos/química , Ribosomas/metabolismoRESUMEN
The present review attempts to deal with movement of tRNA substrates through the peptidyl transferase centre on the large ribosomal subunit and to explain how this movement is interrupted by antibiotics. It builds on the concept of hybrid tRNA states forming on ribosomes and on the observed movement of the 5' end of P-site-bound tRNA relative to the ribosome that occurs on peptide bond formation. The 3' ends of the tRNAs enter, and move through, a catalytic cavity where antibiotics are considered to act by at least three primary mechanisms: (i) they interfere with the entry of the aminoacyl moiety into the catalytic cavity before peptide bond formation; (ii) they inhibit movement of the nascent peptide along the peptide channel, a process that may generally involve destabilization of the peptidyl tRNA, and (iii) they prevent movement of the newly deacylated tRNA between the P/P and hybrid P/E sites on peptide bond formation.
Asunto(s)
Antibacterianos/farmacología , Peptidil Transferasas/química , ARN de Transferencia/efectos de los fármacos , Secuencia de Bases , Sitios de Unión , Catálisis , Datos de Secuencia Molecular , Movimiento (Física) , Conformación de Ácido NucleicoRESUMEN
Random mutations were generated in the lower half of the peptidyl transferase loop in domain V of 23 S rRNA from Escherichia coli using a polymerase chain reaction (PCR) approach, a rapid procedure for identifying mutants and a plasmid-based expression system. The effects of 21 single-site mutations, at 18 different positions, on cell growth, mutant rRNA incorporation into ribosomes and peptidyl transferase activity of the mutant ribosomes were analysed. The general importance of the whole region for the peptidyl transferase centre was emphasized by the finding that 14 of the mutants were sick, or very sick, when ribosomes containing chromosomal-encoded 23 S rRNA were inhibited by erythromycin, and all except one of these exhibited low levels of peptidyl transferase activity in their mutated ribosomes. Two mutations, psi 2580-->C and U2584-->G that both yielded inactive ribosomes were assigned to the donor substrate binding site and a possible base-pairing interaction between the 3'-terminal sequence of the peptidyl-tRNA and the sequence psi/U-G-G2582, that is conserved in all the non-mitochondrial 23 S-like rRNA sequences, is proposed. Three sites that have been implicated in aminoacyl-tRNA binding were mutated: mutant m6A2503G yielded inactive ribosomes, while ribosomes from mutants Um2552A/C and U2555C yielded low and normal activities, respectively. Three mutants, U2528C, G2550A and A2565U, provide evidence for conformational rearrangements occurring in the peptidyl transferase centre which may be affected by the subunit-subunit interaction. Other mutants which yielded ribosomes that were seriously defective in peptidyl transferase activity were U2493A, U2493C, A2497G, A2530G, G2557A and A2589G.