The complex of tmRNA-SmpB and EF-G on translocating ribosomes.

Bacterial ribosomes stalled at the 3' end of malfunctioning messenger RNAs can be rescued by transfer-messenger RNA (tmRNA)-mediated trans-translation. The SmpB protein forms a complex with the tmRNA, and the transfer-RNA-like domain (TLD) of the tmRNA then enters the A site of the ribosome. Subsequently, the TLD-SmpB module is translocated to the P site, a process that is facilitated by the elongation factor EF-G, and translation is switched to the mRNA-like domain (MLD) of the tmRNA. Accurate loading of the MLD into the mRNA path is an unusual initiation mechanism. Despite various snapshots of different ribosome-tmRNA complexes at low to intermediate resolution, it is unclear how the large, highly structured tmRNA is translocated and how the MLD is loaded. Here we present a cryo-electron microscopy reconstruction of a fusidic-acid-stalled ribosomal 70S-tmRNA-SmpB-EF-G complex (carrying both of the large ligands, that is, EF-G and tmRNA) at 8.3 Å resolution. This post-translocational intermediate (TI(POST)) presents the TLD-SmpB module in an intrasubunit ap/P hybrid site and a tRNA(fMet) in an intrasubunit pe/E hybrid site. Conformational changes in the ribosome and tmRNA occur in the intersubunit space and on the solvent side. The key underlying event is a unique extra-large swivel movement of the 30S head, which is crucial for both tmRNA-SmpB translocation and MLD loading, thereby coupling translocation to MLD loading. This mechanism exemplifies the versatile, dynamic nature of the ribosome, and it shows that the conformational modes of the ribosome that normally drive canonical translation can also be used in a modified form to facilitate more complex tasks in specialized non-canonical pathways.

Structural aspects of trans-translation.

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.

Structural features of the tmRNA-ribosome interaction.

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.

One SmpB molecule accompanies tmRNA during its passage through the ribosomes.

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.

Stepping transfer messenger RNA through the ribosome.

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.

How does tmRNA move through the ribosome?

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.