Importance of the conserved nucleotides around the tRNA-like structure of Escherichia coli transfer-messenger RNA for protein tagging

A bacterial RNA functioning as both tRNA ands mRNA, transfer-messenger RNA (tmRNA) rescues stalled ribosomes and clears the cell of incomplete polypep tides. For function, Escherichia coli tmRNA requires an elaborate interplay between a tRNA-like structure and an internal mRNA domain that are connect ed by a 295 nt long compact secondary structure. The tRNA-like structure is surrounded by 16 unpaired nt, including 10 residues that are >95% conserve d among the known 140 tmRNA sequences. All these residues were mutated to d efine their putative role(s) in trans-translation. Both the extent of amino acylation and the alanine incorporation into the tag sequence, reflecting t he two functions of tmRNA, were measured in vitro for all variants. As anti cipated from the low sequence conservation, mutating positions 8-12 and pos ition 15 affects neither aminoacylation nor protein tagging. Mutating a set of two conserved positions 13 and 14 abolishes both functions. Probing the solution conformation indicates that this defective mutant adopts an alter nate conformation of its acceptor stem that is no, more aminoacylatable, an d thus inactive in protein tagging. Selected point mutations at the conserv ed nucleotide stretches 16-20 and 333-335 seriously impair protein tagging with only minor changes: in their solution conformations and aminoacylation . Point mutations at conserved positions 19 and 334 abolish trans-translati on and 70S ribosome binding, although retaining nearly normal aminoacylatio n capacities. Two proteins that are known to interact with tmRNA were purif ied, and their interactions with the defective RNA variants were examined i n vitro. Based on phylogenetic and functional data, an additional structura l motif consisting of a quartet composed of non-Watson-Crick base pairs 5'- YGAC-3':5'-GGAC-3' involving some of the conserved nucleotides next to the tRNA-like portion is proposed. Overall, the highly conserved nucleotides ar ound the tRNA-like portion are maintained for both structural and functiona l requirements during evolution.