RESUMO
In eukaryotes, a crucial step of translation initiation is the binding of the multifactor complex eIF4F to the 5' end of the mRNA, a prerequisite to recruitment of the activated small ribosomal 43S particle. Histone H4 mRNAs have short 5'UTRs, which do not conform to the conventional scanning-initiation model. Here we show that the ORF of histone mRNA contains two structural elements critical for translation initiation. One of the two structures binds eIF4E without the need of the cap. Ribosomal 43S particles become tethered to this site and directly loaded in the vicinity of the AUG. The other structure, 19 nucleotides downstream of the initiation codon, forms a three-way helix junction, which sequesters the m(7)G cap. This element facilitates direct positioning of the ribosome on the cognate start codon. This unusual translation initiation mode might be considered as a hybrid mechanism between the canonical and the IRES-driven translation initiation process.
Assuntos
Histonas/genética , Iniciação Traducional da Cadeia Peptídica/fisiologia , Capuzes de RNA/química , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Sítios de Ligação , Fator de Iniciação 4E em Eucariotos/metabolismo , Histonas/análise , Histonas/química , Luciferases/análise , Camundongos , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Capuzes de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/análise , Ribossomos/fisiologiaRESUMO
Recognition of tRNA by the cognate aminoacyl-tRNA synthetase during translation is crucial to ensure the correct expression of the genetic code. To understand tRNA(Leu) recognition sets and their evolution, the recognition of tRNA(Leu) by the leucyl-tRNA synthetase (LeuRS) from the primitive hyperthermophilic bacterium Aquifex aeolicus was studied by RNA probing and mutagenesis. The results show that the base A73; the core structure of tRNA formed by the tertiary interactions U8-A14, G18-U55 and G19-C56; and the orientation of the variable arm are critical elements for tRNA(Leu) aminoacylation. Although dispensable for aminoacylation, the anticodon arm carries discrete editing determinants that are required for stabilizing the conformation of the post-transfer editing state and for promoting translocation of the tRNA acceptor arm from the synthetic to the editing site.
Assuntos
Leucina-tRNA Ligase/metabolismo , RNA de Transferência de Leucina/química , Aminoacilação de RNA de Transferência , Aminoácidos/química , Anticódon/química , Bactérias/enzimologia , Bactérias/genética , Sequência de Bases , Iodo , Leucina-tRNA Ligase/química , Dados de Sequência Molecular , Mutagênese , Conformação de Ácido Nucleico , Pegadas de Proteínas , RNA de Transferência de Leucina/genética , RNA de Transferência de Leucina/metabolismo , Ribonucleases , Especificidade por SubstratoRESUMO
In metazoans, cell-cycle-dependent histones are produced from poly(A)-lacking mRNAs. The 3' end of histone mRNAs is formed by an endonucleolytic cleavage of longer precursors between a conserved stem-loop structure and a purine-rich histone downstream element (HDE). The cleavage requires at least two trans-acting factors: the stem-loop binding protein (SLBP), which binds to the stem-loop and the U7 snRNP, which anchors to histone pre-mRNAs by annealing to the HDE. Using RNA structure-probing techniques, we determined the secondary structure of the 3'-untranslated region (3'-UTR) of mouse histone pre-mRNAs H4-12, H1t and H2a-614. Surprisingly, the HDE is embedded in hairpin structures and is therefore not easily accessible for U7 snRNP anchoring. Probing of the 3'-UTR in complex with SLBP revealed structural rearrangements leading to an overall opening of the structure especially at the level of the HDE. Electrophoretic mobility shift assays demonstrated that the SLBP-induced opening of HDE actually facilitates U7 snRNA anchoring on the histone H4-12 pre-mRNAs 3' end. These results suggest that initial binding of the SLBP functions in making the HDE more accessible for U7 snRNA anchoring.
Assuntos
Regiões 3' não Traduzidas/química , Histonas/genética , Proteínas Nucleares/metabolismo , Processamento de Terminações 3' de RNA , Precursores de RNA/química , RNA Nuclear Pequeno/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Regiões 3' não Traduzidas/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Humanos , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Pegadas de Proteínas , Precursores de RNA/metabolismoRESUMO
Histone proteins are essential components of eukaryotic chromosomes. In metazoans, they are produced from the so-called replication-dependent histone genes. The biogenesis of histones is tightly coupled to DNA replication in a stoichiometric manner because an excess of histones is highly toxic for the cell. Therefore, a strict cell cycle-regulation of critical factors required for histone expression ensures exclusive S-phase expression. This review focuses on the molecular mechanisms responsible for such a fine expression regulation. Among these, a large part will be dedicated to post-transcriptional events occurring on histone mRNA, like histone mRNA 3' end processing, nucleo-cytoplasmic mRNA export, translation and mRNA degradation. Many factors are involved, including an RNA-binding protein called HBP, also called SLBP (for hairpin- or stem-loop-binding protein) that binds to a conserved hairpin located in the 3' UTR part of histone mRNA. HBP plays a pivotal role in the expression of histone genes since it is necessary for most of the steps of histone mRNA metabolism in the cell. Moreover, the strict S-phase expression pattern of histones is achieved through a fine cell cycle-regulation of HBP. A large part of the discussion will be centered on the critical role of HBP in histone biogenesis.
Assuntos
Replicação do DNA , Regulação da Expressão Gênica , Histonas/genética , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Ciclo Celular , Histonas/biossíntese , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/genética , Fase S , Transcrição GênicaRESUMO
In 1996, a new method, termed the yeast three-hybrid system, dedicated to selection of RNA binding proteins using a hybrid RNA molecule as bait was described. In this minireview, we summarize the results that have been obtained using this method. Indeed, approximately 20 unknown proteins have been characterized so far. The three-hybrid strategy has also been used as a tool to dissect RNA-protein interactions. The example of such a study on human histone HBP interaction with its target mRNA is described. Problems that can be encountered are addressed in a troubleshooting section. Especially, our results with tRNA binding proteins are discussed.
Assuntos
Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido , Histonas/genética , Humanos , RNA/química , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The histone hairpin binding protein (HBP, also called SLBP, which stands for stem-loop binding protein) binds specifically to a highly conserved hairpin structure located in the 3' UTR of the cell-cycle-dependent histone mRNAs. HBP consists of a minimal central RNA binding domain (RBD) flanked by an N- and C-terminal domain. The yeast three-hybrid system has been used to investigate the critical residues of the human HBP involved in the binding of its target hairpin structure. By means of negative selections followed by positive selections, we isolated mutant HBP species. Our results indicate tight relationships between the RBD and the N- and C-terminal domains.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Cromossômicas não Histona , RNA/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Proteínas de Transporte/química , Histonas/genética , Humanos , Óperon Lac/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Nucleotídeos/química , Nucleotídeos/genética , Nucleotídeos/metabolismo , RNA/química , RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Aminoacyl-tRNA synthetases contain one or three Mg(2+) ions in their catalytic sites. In addition to their role in ATP binding, these ions are presumed to play a role in catalysis by increasing the electropositivity of the alpha-phosphate and stabilizing the pentavalent transition state. In the class II aaRS, two highly conserved carboxylate residues have been shown to participate with Mg(2+) ions in binding and coordination. It is shown here that these carboxylate residues are absolutely required for the activity of Saccharomyces cerevisiae aspartyl-tRNA synthetase. Mutants of these residues exhibit pleiotropic effects on the kinetic parameters suggesting an effect at an early stage of the aminoacylation reaction, such as the binding of ATP, Mg(2+), aspartic acid, or the amino acid activation. Despite genetic selections in an APS-knockout yeast strain, we were unable to select a single active mutant of these carboxylate residues. Nevertheless, we isolated an intragenic suppressor from a combinatorial library. The active mutant showed a second substitution close to the first one, and exhibited a significant increase of the tRNA aminoacylation rate. Structural analysis suggests that the acceptor stem of the tRNA might be repositioned to give a more productive enzyme:tRNA complex. Thus, the initial defect of the activation reaction was compensated by a significant increase of the aminoacylation rate that led to cellular complementation.