RESUMO
During 30S ribosomal subunit biogenesis, assembly factors are believed to prevent accumulation of misfolded intermediate states of low free energy that slowly convert into mature 30S subunits, namely, kinetically trapped particles. Among the assembly factors, the circularly permuted GTPase, RsgA, plays a crucial role in the maturation of the 30S decoding center. Here, directed hydroxyl radical probing and single particle cryo-EM are employed to elucidate RsgAÎs mechanism of action. Our results show that RsgA destabilizes the 30S structure, including late binding r-proteins, providing a structural basis for avoiding kinetically trapped assembly intermediates. Moreover, RsgA exploits its distinct GTPase pocket and specific interactions with the 30S to coordinate GTPase activation with the maturation state of the 30S subunit. This coordination validates the architecture of the decoding center and facilitates the timely release of RsgA to control the progression of 30S biogenesis.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , GTP Fosfo-Hidrolases/química , Domínio Catalítico , Microscopia Crioeletrônica , Ativação Enzimática , Proteínas de Escherichia coli/fisiologia , GTP Fosfo-Hidrolases/fisiologia , Guanosina Trifosfato/química , Ligação de Hidrogênio , Hidrólise , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Subunidades Ribossômicas Menores de BactériasRESUMO
The solid-phase DNA probe method is a well-established technique for tRNA purification. We have applied this method for purification and analysis of other non-coding RNAs. Three columns for purification of tRNAPhe, transfer-messenger RNA (tmRNA) and 16S rRNA from Thermus thermophilus were connected in tandem and purifications were performed. From each column, tRNAPhe, tmRNA and 16S rRNA could be purified in a single step. This is the first report of purification of native tmRNA from T. thermophilus and the purification demonstrates that the solid-phase DNA probe method is applicable to non-coding RNA, which is present in lower amounts than tRNA. Furthermore, if a long non-coding RNA is cleaved site-specifically and the fragment can be purified by the solid-phase DNA probe method, modified nucleosides in the long non-coding RNA can be analysed. Therefore, we designed a deoxyribozyme (DNAzyme) to perform site-specific cleavage of 16S rRNA, examined optimum conditions and purified the resulting RNA fragment. Sequencing of complimentary DNA and mass spectrometric analysis revealed that the purified RNA corresponded to the targeted fragment of 16S rRNA. Thus, the combination of DNAzyme cleavage and purification using solid-phase DNA probe methodology can be a useful technique for analysis of modified nucleosides in long non-coding RNAs.
Assuntos
Sondas de DNA , DNA Catalítico/metabolismo , Clivagem do RNA , RNA Bacteriano/isolamento & purificação , RNA Longo não Codificante/análise , RNA Ribossômico 16S/isolamento & purificação , RNA de Transferência/isolamento & purificação , Thermus thermophilus/química , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Nucleosídeos/análise , RNA de Transferência/químicaRESUMO
RadA is involved in strand exchange reactions in homologous recombination which is a fundamental process in all organisms. Sulfolobus tokodaii, one of the archeabacteria, is an aerobic thermoacidophilic crenearchaeon which was isolated from hot springs. RadA from S. tokodaii (stRadA) is a heat resistance protein. The purified protein showed a single band on SDS-PAGE, and gel filtration analysis indicated that stRadA formed a multimeric structure. To reveal a mechanism of the homologous recombination at atomic resolution, stRadA was crystallized with DNA, and its crystals were obtained in 92 conditions by vapor-diffusion methods.