RESUMO
HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme that converts the genomic viral RNA into proviral DNA. Despite intensive biochemical and structural studies, direct thermodynamic data regarding RT interactions with its substrates are still lacking. Here we addressed the mechanism of action of RT and of non-nucleoside RT inhibitors (NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC approach, a step-by-step thermodynamic dissection of the RT polymerization activity showed that most of the driving force for DNA synthesis is provided by initial dNTP binding. Surprisingly, thermodynamic and kinetic data led to a reinterpretation of the mechanism of inhibition of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered by a kinetic barrier and NNRTIs mostly interact with free RT. Once formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent orientation or form high-affinity dead-end complexes, both RT/NNRTI/DNA complexes being unable to bind the incoming nucleotide substrate.
Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Termodinâmica , Calorimetria , Transcriptase Reversa do HIV/química , Nucleotídeos/química , Nucleotídeos/metabolismo , Polimerização , Inibidores da Transcriptase Reversa/química , Relação Estrutura-AtividadeRESUMO
We present a protocol for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. The approach is exemplified by tRNA(Val)-3'-NH-VFLVM-NH(2) and relies on commercially available Escherichia coli tRNA(Val). This tRNA was cleaved site-specifically within the TΨC loop using a 10-23 type DNA enzyme to obtain a 58 nt tRNA 5'-fragment which contained the modifications. After cleavage of the 2',3'-cyclophosphate moiety from the 5'-fragment, it was ligated to the 18 nt RNA-pentapeptide conjugate which had been chemically synthesized. By this methodology, tRNA(Val)-3'-NH-VFLVM-NH(2) is accessible in efficient manner. Furthermore, we point out that the approach is applicable to other types of tRNA.
Assuntos
DNA Catalítico/metabolismo , Farmacorresistência Bacteriana , Macrolídeos/farmacologia , Peptídeos , Estabilidade de RNA , Aminoacil-RNA de Transferência/síntese química , RNA de Transferência de Valina/química , Antibacterianos/farmacologia , Sequência de Bases , Escherichia coli , Espectrometria de Massas , Modelos Moleculares , Conformação de Ácido Nucleico , Fenol/química , Fosforilação , RNA Bacteriano/metabolismo , RNA de Transferência de Valina/síntese química , RNA de Transferência de Valina/isolamento & purificação , RNA de Transferência de Valina/metabolismoRESUMO
RNA-peptide conjugates that mimic amino acid-charged tRNAs and peptidyl-tRNAs are of high importance for structural and functional investigations of ribosomal complexes. Here, we present the synthesis of glycyl-, alanyl-, and isoleucyladenosine modified solid supports that are eligible for the synthesis of stable 3'-aminoacyl- and 3'-peptidyl-tRNA termini with an amide instead of the natural ester linkage. The present work significantly expands the range of accessible peptidyl-tRNA mimics for ribosomal studies.
Assuntos
Poliestirenos/química , Aminoacil-RNA de Transferência/síntese química , RNA/química , Adenosina/química , Alanina/química , Glicina/química , Hidrólise , Isoleucina/química , Peptídeos/química , Aminoacil-RNA de Transferência/química , Ribossomos/metabolismo , Técnicas de Síntese em Fase SólidaAssuntos
Azidas/química , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Animais , Azidas/síntese química , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Desoxiadenosinas/síntese química , Desoxiadenosinas/química , Desoxiadenosinas/genética , Desoxiuridina/análogos & derivados , Desoxiuridina/síntese química , Desoxiuridina/química , RNA Interferente Pequeno/síntese química , Especificidade por SubstratoAssuntos
Azidas/síntese química , Desoxiadenosinas/síntese química , Escherichia coli/química , RNA Bacteriano/síntese química , RNA de Transferência de Metionina/síntese química , Sequência de Bases , Escherichia coli/genética , Hidrólise , RNA Bacteriano/química , RNA de Transferência de Metionina/químicaRESUMO
The 3'-peptidyl-tRNA conjugates that possess a hydrolysis-resistant ribose-3'-amide linkage instead of the natural ester linkage would represent valuable substrates for ribosomal studies. Up to date, access to these derivatives is severely limited. Here, we present a novel approach for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. In short, the approach is based on tRNAs from natural sources that are site-specifically cleaved within the TΨC loop by using DNA enzymes to obtain defined tRNA 5'-fragments carrying the modifications. After dephosphorylation of the 2',3'-cyclophosphate moieties from these fragments, they are ligated to the respective 3'-peptidylamino-tRNA termini that were prepared following the lines of a recently reported solid-phase synthesis. By this novel concept, non-hydrolyzable 3'-peptidyl-tRNA conjugates possessing all natural nucleoside modifications are accessible in highly efficient manner.
Assuntos
Peptídeos/química , RNA de Transferência/química , Sequência de Bases , DNA Catalítico/metabolismo , Hidrólise , Dados de Sequência Molecular , RNA Ligase (ATP)/metabolismo , RNA de Transferência/metabolismoRESUMO
The flatworm stem cell system is exceptional within the animal kingdom, as totipotent stem cells (neoblasts) are the only dividing cells within the organism. In contrast to most organisms, piwi-like gene expression in flatworms is extended from germ cells to somatic stem cells. We describe the isolation and characterization of the piwi homologue macpiwi in the flatworm Macrostomum lignano. We use in situ hybridization, antibody staining and RNA interference to study macpiwi expression and function in adults, during postembryonic development, regeneration and upon starvation. We found novelties regarding piwi function and observed differences to current piwi functions in flatworms. First, macpiwi was essential for the maintenance of somatic stem cells in adult animals. A knock-down of macpiwi led to a complete elimination of stem cells and death of the animals. Second, the regulation of stem cells was different in adults and regenerates compared to postembryonic development. Third, sexual reproduction of M. lignano allowed to follow germline formation during postembryonic development, regeneration, and starvation. Fourth, piwi expression in hatchlings further supports an embryonic formation of the germline in M. lignano. Our findings address new questions in flatworm stem cell research and provide a basis for comparison with higher organisms.
Assuntos
Platelmintos/crescimento & desenvolvimento , Regeneração/fisiologia , Células-Tronco/citologia , Turbelários/crescimento & desenvolvimento , Animais , Diferenciação Celular/fisiologia , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Homeostase , Imuno-Histoquímica , Microscopia Eletrônica , Filogenia , Platelmintos/fisiologia , Interferência de RNA , Células-Tronco/metabolismo , Cauda/fisiologiaRESUMO
Translation of specific small peptides on the ribosome can confer resistance to macrolide antibiotics. To reveal the molecular details of this and related phenomena, stable RNA-peptide conjugates that mimic peptidyl-tRNA would be desirable, especially for ribosome structural biology. A flexible solid-phase synthesis strategy now allows efficient access to these highly requested derivatives without restriction on the RNA and peptide sequences.