An analysis of the requirements for tmRNA function.

Abstract

tmRNA is a small, stable RNA that is found in nearly all eubacterial species. A model for tmRNA function, known as <italic>trans</italic>-translation, has been proposed, in which tmRNA acts as both a tRNA and an mRNA. According to the <italic>trans</italic>-translation model, alanine-charged tmRNA enters ribosomes that have become stalled upon reaching the 3<super>'</super> terminus of an mRNA without encountering an in-frame translational stop codon. The alanine is transferred from tmRNA to the nascent peptide, followed by release of the original mRNA from the ribosome. tmRNA then becomes the template for translation, and a 10 amino acid tag sequence is translated, followed by dissociation of the translational ternary complex upon reaching a stop codon. tmRNA-tagged peptides, which now carry a C-terminal protease recognition sequence, are then degraded. <italic>Escherichia coli</italic> strains lacking tmRNA are viable, but exhibit several phenotypes, including the failure to support growth of some bacteriophages. This study examines the requirements for tmRNA function both in the support of bacteriophage growth and for tmRNA-mediated tagging. I begin by assessing whether different mutant forms of tmRNA are active. I find that mutant tmRNAs that cannot be charged with alanine are not active, but that mutant tmRNAs that encode a tag sequence lacking the protease recognition site are active, in supporting growth of 3 different bacteriophages whose growth in <italic> Escherichia coli</italic> requires tmRNA activity. A small protein, SmpB, is identified that is required for all activities of tmRNA that were tested, both in <italic>E. coli</italic> and in <italic>Neisseria gonorrhoeae</italic>, and evidence that species specificity for the putative tmRNA/SmpB complex exists between bacterial species is presented. Finally, An <italic>E. coli </italic> polymorphism, one allele of which suppresses the effects of the absence of tmRNA activity upon both phage and bacterial growth, is characterized and mapped to a region of the <italic>E. coli</italic> chromosome heavily populated by genes encoding ribosomal proteins. The results of the work described in this dissertation are not consistent with the targeting of truncated proteins toward proteolysis being the primary function of tmRNA, as was originally proposed in the <italic>trans</italic>-translation model. Rather, a new model is presented in which the primary function of tmRNA is the removal of stalled ribosomes from intact mRNA as a mechanism for increasing translational efficiency.