RESUMEN
trans-Translation is a process which the bacterial cells apply to rescue the ribosomes that are arrested during the translation of damaged mRNA and to get rid of the mRNA and the product polypeptide. In the course of trans-translation, the mRNA-like domain of tmRNA replaces the nonstop messenger RNA bound to the ribosome. Although several structural elements of tmRNA and SmpB known to be essential for correct determination of resume codon, the molecular mechanism of trans-translation is not well understood. Computer modeling has been used to develop a model for the spatial organization of the tmRNA inside the ribosome at different stages of trans-translation leading to a proposal for the mechanism of the template-switching process.
Asunto(s)
ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Codón/metabolismo , Escherichia coli/metabolismo , Terminación de la Cadena Péptídica Traduccional/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.
Asunto(s)
Escherichia coli/química , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN Mensajero/química , ARN de Transferencia/química , Ribosomas/química , Secuencia de Bases , Microscopía por Crioelectrón , Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Biosíntesis de Proteínas , ARN Bacteriano/metabolismo , ARN Bacteriano/ultraestructura , ARN Mensajero/metabolismo , ARN Mensajero/ultraestructura , ARN de Transferencia/metabolismo , ARN de Transferencia/ultraestructura , Ribosomas/metabolismo , Ribosomas/ultraestructuraRESUMEN
tmRNA and SmpB are the main participants of trans-translation, a process which rescues the ribosome blocked during translation of non-stop mRNA. While a one-to-one stoichiometry of tmRNA to the ribosome is generally accepted, the number of SmpB molecules in the complex is still under question. We have isolated tmRNA-ribosome complexes blocked at different steps of the tmRNA path through the ribosome and analyzed the stoichiometry of the complexes. Ribosome, tmRNA and SmpB were found in equimolar amount in the tmRNA-ribosome complexes stopped at the position of the 2nd, 4th, 5th or the 11th codons of the coding part of the tmRNA.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Biosíntesis de Proteínas , ARN Bacteriano/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN Bacteriano/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
tmRNA (transfer messenger RNA) is a unique molecule used by all bacteria to rescue stalled ribosomes and to mark unfinished peptides with a specific degradation signal. tmRNA is recruited by arrested ribosomes in which it facilitates the translational switch from cellular mRNA to the mRNA part of tmRNA. Small protein B (SmpB) is a key partner for the trans-translation activity of tmRNA both in vivo and in vitro. It was shown that SmpB acts at the initiation step of the trans-translation process by facilitating tmRNA aminoacylation and binding to the ribosome. Little is known about the subsequent steps of trans-translation. Here we demonstrated the first example of an investigation of tmRNA.ribosome complexes at different stages of trans-translation. Our results show that the structural element at the position of tmRNA pseudoknot 3 remains intact during the translation of the mRNA module of tmRNA and that it is localized on the surface of the ribosome. At least one SmpB molecule remains bound to a ribosome.tmRNA complex isolated from the cell when translation is blocked at different positions within the mRNA part of tmRNA.
Asunto(s)
ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN de Transferencia/genéticaRESUMEN
To test the structure of tmRNA in solution, cross-linking experiments were performed which showed two sets of cross-links in two different domains of tmRNA. Site-directed mutagenesis was used to search for tmRNA nucleotide bases that might form a functional analogue of a codon-anticodon duplex to be recognized by the ribosomal A-site. We demonstrate that nucleotide residues U85 and A86 from tmRNA are significant for tmRNA function and propose that they are involved in formation of a tmRNA element playing a central role in A-site recognition. These data are discussed in the frame of a hypothetical model that suggests a general scheme for the interaction of tmRNA with the ribosome and explains how it moves through the ribosome.