| Bacterial cells employ a mechanism known as trans-translation to rescue ribosomes stalled at the 3′-end of broken mRNA. Trans-translation is orchestrated by a small RNA molecule known as tmRNA. The Escherichia coli tmRNA of 363-nucleotides has a tRNA-like structure, a mRNA-like region (tag-peptide coding region), and four pseudoknots (pk1 to pk4). The trans-translation process also requires several proteins, including ribosomal protein S1. The full-length tmRNA binds to the S1 protein, but a mutant lacking 90 to 299 nucleotide residues comprising the tag region and pk2 to pk4 does not. The aim of this project was to determine the regions in the E. coli tmRNA required for binding of protein S1. Fourteen deletion mutants of trnRNA (M1 to M14) involving all possible combinations of tag, pk2, pk3 and pk4 were synthesized and tested for their binding using gel mobility-shift assays. No single tmRNA feature was sufficient to allow complete binding of protein S1. Pair-wise and competition experiments showed that pk4 was the least important region. A truncated version of ribosomal protein S1 (ΔS1) missing the first two of the six domains was expressed, purified and found to be incapable of binding to wild-type tmRNA. The results suggest that a large portion of tmRNA is in contact with numerous domains of ribosorfial protein S1. |
