In eubacterial translation, lack of a stop codon on the mRNA results in a defective, potentially toxic polypeptide stalled on the ribosome. Bacteria possess a specialized mRNA, called transfer messenger RNA (tmRNA), to rescue such a stalled system. tmRNA contains a transfer RNA (tRNA)-like domain (TLD), which enters the ribosome as a tRNA and places an ORF into the mRNA channel. This ORF codes for a signal marking the polypeptide for degradation and ends in a stop codon, leading to release of the faulty polypeptide and recycling of the ribosome. The binding of tmRNA to the stalled ribosome is mediated by small protein B (SmpB). By means of cryo-EM, we obtained a density map for the preaccommodated state of the tmRNA⅐SmpB⅐EF-Tu⅐70S ribosome complex with much improved definition for the tmRNA-SmpB complex, showing two SmpB molecules bound per ribosome, one toward the A site on the 30S subunit side and the other bound to the 50S subunit near the GTPase-associated center. tmRNA is strongly attached to the 30S subunit head by multiple contact sites, involving most of its pseudoknots and helices. The map clarifies that the TLD is located near helix 34 and protein S19 of the 30S subunit, rather than in the A site as tRNA for normal translation, so that the TLD is oriented toward the ORF. elongation factor Tu ͉ preaccommodated state ͉ rescue mechanism ͉ transtranslation ͉ transfer RNA-like domain

Bacterial ribosomes stalled on defective messenger RNAs (mRNAs) are rescued by tmRNA, an approximately 300-nucleotide-long molecule that functions as both transfer RNA (tRNA) and mRNA. Translation then switches from the defective message to a short open reading frame on tmRNA that tags the defective nascent peptide chain for degradation. However, the mechanism by which tmRNA can enter and move through the ribosome is unknown. We present a cryo-electron microscopy study at approximately 13 to 15 angstroms of the entry of tmRNA into the ribosome. The structure reveals how tmRNA could move through the ribosome despite its complicated topology and also suggests roles for proteins S1 and SmpB in the function of tmRNA.

A eubacterial ribosome stalled on a defective mRNA can be released through a quality control mechanism referred to as trans-translation, which depends on the coordinating binding actions of transfer-messenger RNA, small protein B, and ribosome protein S1. By means of cryo-electron microscopy, we obtained a map of the complex composed of a stalled ribosome and small protein B, which appears near the decoding center. This result suggests that, when lacking a codon, the A-site on the small subunit is a target for small protein B. To investigate the role of S1 played in trans-translation, we obtained a cryo-electron microscopic map, including a stalled ribosome, transfermessenger RNA, and small protein Bs but in the absence of S1. In this complex, several connections between the 30 S subunit and transfer-messenger RNA that appear in the ؉S1 complex are no longer found. We propose the unifying concept of scaffolding for the roles of small protein B and S1 in binding of transfer-messenger RNA to the ribosome during trans-translation, and we infer a pathway of sequential binding events in the initial phase of trans-translation.