tmRNA has a dual function both as a tRNA and an mRNA to facilitate trans-translation, in which a ribosome can switch translation from a truncated or other problematic mRNA to the tmRNA's tag-encoding sequence. During trans-translation, tmRNA enters the ribosomal A-site without a codon-anticodon interaction, but with an SmpB which is a tmRNA binding protein. To further study the interaction of SmpB with ribosome and tmRNA, we have performed directed hydroxyl radical probing. It revealed that there are two SmpB binding sites at the A-site and P-site, and their C-terminal regions reside along the mRNA path in the 30S subunit. From these results, we propose a new type of molecular mimicry, in which both tmRNA and SmpB mimic the structures and functions of tRNA and mRNA during trans-translation, addressing how tmRNA preferentially recognizes the stalled ribosome, and what substitutes for a codon-anticodon interaction.