Cryo-EM visualization of transfer messenger RNA with two SmpBs in a stalled ribosome

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 tmRNA.SmpB.EF-Tu.70S 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.