10Sa RNA is a small stable bacterial RNA in which the 5'- and 3'-end sequences are folded into a tRNA-like structure. The RNA is aminoacylatable with alanine in vitro, and it interacts with 70S ribosomes in the cell. Recently, Escherichia coli 10Sa RNA has been shown to contain the sequence-encoding tag-peptides, which are found to attach to the C-termini of truncated peptides synthesized in vivo. We have found that the E coli 10Sa RNA stimulates incorporation of the tag-specific amino acids into proteins depending on the poly(U)-directed poly-phenylalanine synthesis in the in vitro translation system. Our finding supports the 'trans-translation' model proposed for the tag synthesis, in which alanyl-10Sa RNA enters the ribosome when translation stops at the 3'-end of the truncated mRNA lacking a stop codon, and translation of the tag peptide occurs by switching the template from mRNA to 10Sa RNA. In this unique reaction, 10Sa RNA acts both as a tRNA and as an mRNA.