RESUMO
The complete nucleotide sequence of the genomic RNA of the new virus Nemesia ring necrosis virus (NeRNV), which is widespread in various ornamental plant species belonging to the Scrophulariaceae and Verbenaceae, has been determined. Based on its gene content, the folding properties of its 5'-untranslated region and in vitro translation experiments, NeRNV RNA is a typical tymovirus RNA. Its 3' end, however, differs greatly from those of the valine-specific tymoviral RNAs that have been analysed previously. It can be folded into an upstream pseudoknot domain and a histidine-specific tRNA-like structure, a combination that, so far, has been found only in tobamoviral RNAs. The identity elements found in NeRNV RNA for recognition by yeast histidyl-tRNA synthetase are more similar to those of yeast tRNAHis than the ones found in tobacco mosaic virus RNA. As a result NeRNV RNA can be charged with histidine even more efficiently than tobacco mosaic virus RNA.
Assuntos
RNA Viral/genética , Tymovirus/genética , Região 3'-Flanqueadora , Regiões 3' não Traduzidas , Sequência de Bases , Histidina , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Scrophulariaceae/virologia , Alinhamento de Sequência , Especificidade da Espécie , Tobamovirus/genética , Tymovirus/química , Verbenaceae/virologiaRESUMO
Transfer-messenger RNA (tmRNA) mimics functions of aminoacyl-tRNA and mRNA, subsequently, when rescuing stalled ribosomes on a 3' truncated mRNA without stop codon in bacteria. In addition, this mechanism marks prematurely terminated proteins by a C-terminal peptide tag as a signal for degradation by specific cellular proteases. For Escherichia coli, previous studies on initial steps of this "trans-translation" mechanism revealed that tmRNA alanylation by Ala-tRNA synthetase and binding of Ala-tmRNA by EF-Tu-GTP for subsequent delivery to stalled ribosomes are inefficient when compared to analogous reactions with canonical tRNA(Ala). In other studies, protein SmpB and ribosomal protein S1 appeared to bind directly to tmRNA and to be indispensable for trans-translation. Here, we have searched for additional and synergistic effects of the latter two on tmRNA alanylation and its subsequent binding to EF-Tu-GTP. Kinetic analysis of functioning combined with band-shift experiments and structural probing demonstrate, that tmRNA may indeed form a multimeric complex with SmpB, S1 and EF-Tu-GTP, which leads to a considerably enhanced efficiency of the initial steps of trans-translation. Whereas S1 binds to the mRNA region of tmRNA, we have found that SmpB and EF-Tu both interact with its acceptor arm region. Interaction with SmpB and EF-Tu was also observed at the acceptor arm of Ala-tRNA(Ala), but there the alanylation efficiency was inhibited rather than stimulated by SmpB. Therefore, SmpB may function as an essential modulator of the tRNA-like acceptor arm of tmRNA during its successive steps in trans-translation.
Assuntos
Alanina/genética , Escherichia coli/genética , Guanosina Trifosfato/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/metabolismo , Acilação , Alanina/metabolismo , Alanina-tRNA Ligase/metabolismo , Sequência de Bases , Sítios de Ligação , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Cinética , Dados de Sequência Molecular , Ensaios de Proteção de Nucleases , Conformação de Ácido Nucleico , Ligação Proteica , RNA Bacteriano/genética , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Ribonucleases/metabolismo , Proteínas Ribossômicas/metabolismoRESUMO
Aminoacylation and transportation of tmRNA to stalled ribosomes constitute prerequisite steps for trans-translation, a process facilitating the release of stalled ribosomes from 3' ends of truncated mRNAs and the degradation of incompletely synthesized proteins. Kinetic analysis of the aminoacylation of tmRNA indicates that tmRNA has both a lower affinity and a lower turnover number than cognate tRNA(Ala) for alanyl-tRNA synthetase, resulting in a 75-fold lower k(cat)/K(M) value. The association rate constant of Ala-tmRNA for elongation factor Tu in complex with GTP is about 150-fold lower than that of Ala-tRNA(Ala), whereas its dissocation rate constant is about 5-fold lower. These observations can be interpreted to suggest that additional factors facilitate tmRNA binding to ribosomes.