RESUMO
Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2. Biochemical and co-crystallization analyses demonstrate that A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.
Assuntos
Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Linhagem Celular Tumoral , Cristalografia por Raios X , Reparo do DNA/efeitos dos fármacos , Inibidores Enzimáticos/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação/efeitos dos fármacos , Modelos Moleculares , Estrutura MolecularRESUMO
Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.
Assuntos
Antineoplásicos/farmacologia , Indanos/farmacologia , Complexo Repressor Polycomb 2/antagonistas & inibidores , Sulfonamidas/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indanos/química , Modelos Moleculares , Estrutura Molecular , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfonamidas/química , Células Tumorais CultivadasRESUMO
Translational control is frequently exerted at the stage of mRNA recruitment to the initiating ribosome. We have reconstituted mRNA recruitment to the 43S preinitiation complex (PIC) using purified S. cerevisiae components. We show that eIF3 and the eIF4 factors not only stabilize binding of mRNA to the PIC, they also dramatically increase the rate of recruitment. Although capped mRNAs require eIF3 and the eIF4 factors for efficient recruitment to the PIC, uncapped mRNAs can be recruited in the presence of eIF3 alone. The cap strongly inhibits this alternative recruitment pathway, imposing a requirement for the eIF4 factors for rapid and stable binding of natural mRNA. Our data suggest that the 5' cap serves as both a positive and negative element in mRNA recruitment, promoting initiation in the presence of the canonical group of mRNA handling factors while preventing binding to the ribosome via an aberrant, alternative pathway requiring only eIF3.
Assuntos
Regulação Fúngica da Expressão Gênica , Guanosina/análogos & derivados , Iniciação Traducional da Cadeia Peptídica/fisiologia , Capuzes de RNA/metabolismo , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sistema Livre de Células/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Guanosina/metabolismo , Cinética , Conformação de Ácido Nucleico , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Análogos de Capuz de RNA/fisiologia , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Herein we disclose SAR studies of a series of dimethylamino pyrrolidines which we recently reported as novel inhibitors of the PRC2 complex through disruption of EED/H3K27me3 binding. Modification of the indole and benzyl moieties of screening hit 1 provided analogs with substantially improved binding and cellular activities. This work culminated in the identification of compound 2, our nanomolar proof-of-concept (PoC) inhibitor which provided on-target tumor growth inhibition in a mouse xenograft model. X-ray crystal structures of several inhibitors bound in the EED active-site are also discussed.
Assuntos
Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/metabolismo , Pirrolidinas/farmacologia , Sulfonamidas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Ligantes , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Complexo Repressor Polycomb 2/química , Ligação Proteica , Pirrolidinas/síntese química , Pirrolidinas/química , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Herein we disclose SAR studies that led to a series of isoindoline ureas which we recently reported were first-in-class, non-substrate nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. Modification of the isoindoline and/or the terminal functionality of screening hit 5 provided inhibitors such as 52 and 58 with nanomolar antiproliferative activity and preclinical pharmacokinetics properties which enabled potent antitumor activity when dosed orally in mouse xenograft models. X-ray crystal structures of two inhibitors bound in the NAMPT active-site are discussed.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Citocinas/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Citocinas/química , Citocinas/metabolismo , Descoberta de Drogas , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Humanos , Isoindóis/química , Isoindóis/farmacocinética , Isoindóis/farmacologia , Isoindóis/uso terapêutico , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Nicotinamida Fosforribosiltransferase/química , Nicotinamida Fosforribosiltransferase/metabolismo , Relação Estrutura-Atividade , Ureia/farmacocinética , Ureia/uso terapêuticoRESUMO
Most microbes have not been cultured, and many of those that are cultivatable are difficult, dangerous or expensive to propagate or are genetically intractable. Routine cloning of large genome fractions or whole genomes from these organisms would significantly enhance their discovery and genetic and functional characterization. Here we report the cloning of whole bacterial genomes in the yeast Saccharomyces cerevisiae as single-DNA molecules. We cloned the genomes of Mycoplasma genitalium (0.6 Mb), M. pneumoniae (0.8 Mb) and M. mycoides subspecies capri (1.1 Mb) as yeast circular centromeric plasmids. These genomes appear to be stably maintained in a host that has efficient, well-established methods for DNA manipulation.
Assuntos
Clonagem Molecular/métodos , Genoma Bacteriano , Mycoplasma/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Diploide , Vetores Genéticos/química , Dados de Sequência Molecular , Mycoplasma genitalium/genética , Mycoplasma mycoides/genética , Mycoplasma pneumoniae/genética , Recombinação GenéticaRESUMO
We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments.
Assuntos
DNA/biossíntese , Genoma Bacteriano/genética , Mycoplasma genitalium/genética , Oligodesoxirribonucleotídeos/genética , Leveduras/genética , Clonagem Molecular/métodos , Oligodesoxirribonucleotídeos/metabolismo , Recombinação GenéticaRESUMO
Over the past several years, significant advances have been made in the molecular genetics of the Mollicutes (the simplest cells that can be grown in axenic culture). Nevertheless, a number of basic molecular tools are still required before genetic manipulations become routine. Here we describe the development of a new dominant selectable marker based on the enzyme puromycin-N-acetyltransferase from Streptomyces alboniger. Puromycin is an antibiotic that mimics the 3'-terminal end of aminoacylated tRNAs and attaches to the carboxyl terminus of growing protein chains. This stops protein synthesis. Because puromycin conscripts rRNA recognition elements that are used by all of the various tRNAs in a cell, it is unlikely that spontaneous antibiotic resistance can be acquired via a simple point mutation--an annoying issue with existing mycoplasma markers. Our codon-optimized cassette confers pronounced puromycin resistance on all five of the mycoplasma species we have tested so far. The resistance cassette was also designed to function in Escherichia coli, which simplifies the construction of shuttle vectors and makes it trivial to produce the large quantities of DNA generally necessary for mycoplasma transformation. Due to these and other features, we expect the puromycin marker to be a widely applicable tool for studying these simple cells and pathogens.
Assuntos
Genoma Bacteriano/genética , Mycoplasma/genética , Acetiltransferases/genética , Antibacterianos/farmacologia , Modelos Genéticos , Mycoplasma/efeitos dos fármacos , Puromicina/farmacologiaRESUMO
PARP inhibitors have recently been approved as monotherapies for the treatment of recurrent ovarian cancer and metastatic BRCA-associated breast cancer, and ongoing studies are exploring additional indications and combinations with other agents. PARP inhibitors trap PARP onto damaged chromatin when combined with temozolomide and methyl methanesulfonate, but the clinical relevance of these findings remains unknown. PARP trapping has thus far been undetectable in cancer cells treated with PARP inhibitors alone. Here, we evaluate the contribution of PARP trapping to the tolerability and efficacy of PARP inhibitors in the monotherapy setting. We developed a novel implementation of the proximity ligation assay to detect chromatin-trapped PARP1 at single-cell resolution with higher sensitivity and throughput than previously reported methods. We further demonstrate that the PARP inhibitor-induced trapping appears to drive single-agent cytotoxicity in healthy human bone marrow, indicating that the toxicity of trapped PARP complexes is not restricted to cancer cells with homologous recombination deficiency. Finally, we show that PARP inhibitors with dramatically different trapping potencies exhibit comparable tumor growth inhibition at MTDs in a xenograft model of BRCA1-mutant triple-negative breast cancer. These results are consistent with emerging clinical data and suggest that the inverse relationship between trapping potency and tolerability may limit the potential therapeutic advantage of potent trapping activity. IMPLICATIONS: PARP trapping contributes to single-agent cytotoxicity of PARP inhibitors in both cancer cells and healthy bone marrow, and the therapeutic advantage of potent trapping activity appears to be limited.
Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Animais , Medula Óssea , Citotoxicidade Imunológica , Feminino , Humanos , Camundongos , Camundongos SCID , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologiaRESUMO
Selecting the codon at which to begin translation is a complicated event in an already complicated process. Many protein initiation factors (eIFs) have been implicated in start site selection, but the mechanistic details of their activities have remained obscure until recently. Biochemical and genetic studies of eIFs 1, 1A, 2 and 5 have suggested that the 43S pre-initiation complex exists in two conformations and that the changing interactions of the factors within the 43S pre-initiation complex in response to encountering an AUG codon regulates these conformations and, ultimately, the selection of the start codon.
Assuntos
Códon de Iniciação , Células Eucarióticas/fisiologia , Fatores de Terminação de Peptídeos/fisiologia , Transcrição Gênica , Animais , Humanos , Modelos Biológicos , Conformação ProteicaRESUMO
During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex scans the mRNA in search of an AUG codon at which to begin translation. Start codon recognition halts scanning and triggers a number of events that commit the complex to beginning translation at that point on the mRNA. Previous studies in vitro and in vivo have indicated that eukaryotic initiation factors (eIFs) 1, 2 and 5 play key roles in these events. In addition, it was reported recently that the C-terminal domain of eIF1A is involved in maintaining the fidelity of start codon recognition. The molecular mechanisms by which these factors work together to ensure fidelity of start site selection remain poorly understood. Here, we report the quantitative characterization of energetic interactions between eIF1A, eIF5 and the AUG codon in an in vitro reconstituted yeast translation initiation system. Our results show that recognition of an AUG codon by the 43 S complex triggers an interaction between eIF5 and eIF1A, resulting in a shift in the equilibrium between two states of the pre-initiation complex. This AUG-dependent change may be a reorganization from a scanning-competent state to a scanning-incompetent state. Mutations in both eIF1A and eIF5 that increase initiation at non-AUG codons in vivo weaken the interaction between the two factors upon AUG recognition, while specifically strengthening it in response to a UUG codon. These data suggest strongly that the interaction between eIF1A and eIF5 is involved in maintaining the fidelity of start codon recognition in vivo.
Assuntos
Códon de Iniciação/química , Fator de Iniciação 1 em Eucariotos/química , Fator de Iniciação 5 em Eucariotos/química , Metionina/química , Ribossomos/química , Proteínas de Saccharomyces cerevisiae/química , Transcrição Gênica , Códon de Iniciação/metabolismo , Fator de Iniciação 1 em Eucariotos/genética , Fator de Iniciação 1 em Eucariotos/fisiologia , Fator de Iniciação 5 em Eucariotos/fisiologia , Metionina/metabolismo , Mutação Puntual , Biossíntese de Proteínas/genética , Estrutura Terciária de Proteína , Precursores de RNA/genética , Precursores de RNA/metabolismo , Ribossomos/genética , Ribossomos/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , TermodinâmicaRESUMO
Many methods have been developed for the cloning of PCR products. These methods include blunt-end cloning, TA cloning, and using restriction sites incorporated into the PCR primers. The restrictionless cloning technique allows efficient directional cloning of PCR products into any cloning site within a vector regardless of whether the sites are contained within the insert to be cloned.
Assuntos
Clonagem Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Primers do DNA/genética , Escherichia coli/genética , Vetores GenéticosRESUMO
During the 2009 H1N1 influenza pandemic, vaccines for the virus became available in large quantities only after human infections peaked. To accelerate vaccine availability for future pandemics, we developed a synthetic approach that very rapidly generated vaccine viruses from sequence data. Beginning with hemagglutinin (HA) and neuraminidase (NA) gene sequences, we combined an enzymatic, cell-free gene assembly technique with enzymatic error correction to allow rapid, accurate gene synthesis. We then used these synthetic HA and NA genes to transfect Madin-Darby canine kidney (MDCK) cells that were qualified for vaccine manufacture with viral RNA expression constructs encoding HA and NA and plasmid DNAs encoding viral backbone genes. Viruses for use in vaccines were rescued from these MDCK cells. We performed this rescue with improved vaccine virus backbones, increasing the yield of the essential vaccine antigen, HA. Generation of synthetic vaccine seeds, together with more efficient vaccine release assays, would accelerate responses to influenza pandemics through a system of instantaneous electronic data exchange followed by real-time, geographically dispersed vaccine production.
Assuntos
Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Pandemias/prevenção & controle , Vacinas Sintéticas/imunologia , Animais , Linhagem Celular , Simulação por Computador , Cães , Genes Sintéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Subtipo H7N9 do Vírus da Influenza A/imunologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Neuraminidase/genética , Vírus Reordenados/imunologia , Reprodutibilidade dos Testes , Carga ViralRESUMO
The sequenced genome of Mycoplasma mycoides subsp. capri revealed the presence of a Type III restriction-modification system (MmyCI). The methyltransferase (modification) subunit of MmyCI (M.MmyCI) was shown to recognize the sequence 5'-TGAG-3' and methylate the adenine. The coding region of the methyltransferase gene contains 12 consecutive AG dinucleotide repeats that result in a translational termination at a TAA codon immediately beyond the repeat region. This strain does not have MmyCI activity. A clone was found with 10 AG repeats such that the gene is in frame, and this strain has MmyCI activity, suggesting that the expression of the MmyCI methyltransferase may be phase variable.
Assuntos
Proteínas de Bactérias/metabolismo , Enzimas de Restrição-Modificação do DNA/metabolismo , DNA Bacteriano/metabolismo , Repetições de Dinucleotídeos/fisiologia , Mycoplasma mycoides/enzimologia , Proteínas de Bactérias/genética , Enzimas de Restrição-Modificação do DNA/genética , DNA Bacteriano/genética , Mycoplasma mycoides/genética , Especificidade por Substrato/fisiologiaRESUMO
We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.
Assuntos
Bioengenharia , Engenharia Genética , Genoma Bacteriano , Mycoplasma capricolum/genética , Mycoplasma mycoides/genética , Proteínas de Bactérias/análise , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/síntese química , DNA Bacteriano/genética , Escherichia coli/genética , Deleção de Genes , Genes Bacterianos , Dados de Sequência Molecular , Mycoplasma mycoides/crescimento & desenvolvimento , Mycoplasma mycoides/fisiologia , Mycoplasma mycoides/ultraestrutura , Fenótipo , Plasmídeos , Reação em Cadeia da Polimerase , Polimorfismo Genético , Saccharomyces cerevisiae/genética , Transformação BacterianaRESUMO
We recently reported the chemical synthesis, assembly, and cloning of a bacterial genome in yeast. To produce a synthetic cell, the genome must be transferred from yeast to a receptive cytoplasm. Here we describe methods to accomplish this. We cloned a Mycoplasma mycoides genome as a yeast centromeric plasmid and then transplanted it into Mycoplasma capricolum to produce a viable M. mycoides cell. While in yeast, the genome was altered by using yeast genetic systems and then transplanted to produce a new strain of M. mycoides. These methods allow the construction of strains that could not be produced with genetic tools available for this bacterium.
Assuntos
Clonagem Molecular , Técnicas de Transferência de Genes , Engenharia Genética , Genoma Bacteriano , Mycoplasma capricolum/genética , Mycoplasma mycoides/genética , Saccharomyces cerevisiae/genética , Centrômero , Metilação de DNA , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Mycoplasma mycoides/crescimento & desenvolvimento , Mycoplasma mycoides/isolamento & purificação , Plasmídeos , Análise de Sequência de DNA , Deleção de Sequência , Transformação BacterianaRESUMO
We have synthesized a 582,970-base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.
Assuntos
Clonagem Molecular , DNA Bacteriano/síntese química , Genoma Bacteriano , Genômica/métodos , Mycoplasma genitalium/genética , Sequência de Bases , Cromossomos Artificiais Bacterianos , Cromossomos Artificiais de Levedura , DNA Recombinante , Escherichia coli/genética , Vetores Genéticos , Oligodesoxirribonucleotídeos/síntese química , Plasmídeos , Recombinação Genética , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Transformação GenéticaRESUMO
Initiation of translation is the process by which initiator tRNA and the start codon of mRNA are positioned in the ribosomal P site. In eukaryotes, one of the first steps involves the binding of two small factors, eIF1 and eIF1A, to the small (40S) ribosomal subunit. This facilitates tRNA binding, allows scanning of mRNA, and maintains fidelity of start codon recognition. Using cryo-EM, we have obtained 3D reconstructions of 40S bound to both eIF1 and eIF1A, and with each factor alone. These structures reveal that together, eIF1 and eIF1A stabilize a conformational change that opens the mRNA binding channel. Biochemical data reveal that both factors accelerate the rate of ternary complex (eIF2*GTP*Met-tRNA(i)(Met)) binding to 40S but only eIF1A stabilizes this interaction. Our results suggest that eIF1 and eIF1A promote an open, scanning-competent preinitiation complex that closes upon start codon recognition and eIF1 release to stabilize ternary complex binding and clamp down on mRNA.
Assuntos
Fator de Iniciação 1 em Eucariotos/química , Conformação Molecular , Biossíntese de Proteínas , Ribossomos/química , Saccharomyces cerevisiae/genética , Sítios de Ligação , Microscopia Crioeletrônica , Fator de Iniciação 1 em Eucariotos/genética , Fator de Iniciação 1 em Eucariotos/metabolismo , Modelos Genéticos , Ligação Proteica , RNA Mensageiro/química , RNA Ribossômico/química , Proteínas Ribossômicas/química , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-AtividadeRESUMO
Translation initiation factor eIF1A stimulates preinitiation complex (PIC) assembly and scanning, but the molecular mechanisms of its functions are not understood. We show that the F131A,F133A mutation in the C-terminal tail (CTT) of eIF1A impairs recruitment of the eIF2-GTP-Met-tRNA(i)(Met) ternary complex to 40S subunits, eliminating functional coupling with eIF1. Mutating residues 17-21 in the N-terminal tail (NTT) of eIF1A also reduces PIC assembly, but in a manner rescued by eIF1. Interestingly, the 131,133 CTT mutation enhances initiation at UUG codons (Sui(-) phenotype) and decreases leaky scanning at AUG, while the NTT mutation 17-21 suppresses the Sui(-) phenotypes of eIF5 and eIF2beta mutations and increases leaky scanning. These findings and the opposite effects of the mutations on eIF1A binding to reconstituted PICs suggest that the NTT mutations promote an open, scanning-conducive conformation of the PIC, whereas the CTT mutations 131,133 have the reverse effect. We conclude that tight binding of eIF1A to the PIC is an important determinant of AUG selection and is modulated in opposite directions by residues in the NTT and CTT of eIF1A.