Analysis of the role of trans-translation in the requirement of tmRNA for lambda imm(P22) growth in Escherichia coli

The small, stable RNA molecule encoded by ssrA, known as tmRNA or 10Sa RNA, is required for the growth of certain hybrid lambda imm(P22) phages in Esc herichia coil. tmRNA has been shown to tag partially synthesized proteins f or degradation in vivo by attaching a short peptide sequence, encoded by tm RNA to the carboxyl termini of these proteins. This tag sequence contains, at its C terminus, an amino acid sequence that is recognized by cellular pr oteases and leads to degradation of tagged proteins. A model describing thi s function of tmRNA, the trans-translation model (K. C. Keiler, P. R. Walle r, and R. T. Sauer, Science 271:990-993, 1996), proposes that tmRNA acts fi rst as a tRNA and then as a mRNA, resulting in release of the original mRNA template from the ribosome and translocation of the nascent peptide to tmR NA. Previous work from this laboratory suggested that tmRNA may also intera ct specifically with DNA-binding proteins, modulating their activity. Howev er, more recent results indicate that interactions between tmRNA and DNA-bi nding proteins are likely nonspecific. In light of this new information, we examine the effects on lambda imm(P22) growth of mutations eliminating act ivities postulated to be important for two different steps in the trans-tra nslation model, alanine charging of tmRNA and degradation of tagged protein s. This mutational analysis suggests that, while charging of tmRNA with ala nine is essential for lambda imm(P22) growth in E. coil, degradation of pro teins tagged by tmRNA is required only to achieve optimal levels of phage g rowth. Based on these results, we propose that trans-translation may have t wo roles, the primary role being the release of stalled ribosomes from thei r mRNA template and the secondary role being the tagging of truncated prote ins for degradation.