Trans-translation exposed: understanding the structures and functions of tmRNA-SmpB

Variants of lambda repressor and cytochrome b562 translated from messenger RNAs without stop codons were modified by carboxyl terminal addition of an ssrA-encoded peptide tag and subsequently degraded by carboxyl terminal-specific proteases present in both the cytoplasm and periplasm of Escherichia coli. The tag appears to be added to the carboxyl terminus of the nascent polypeptide chain by cotranslational switching of the ribosome from the damaged messenger RNA to ssrA RNA.

Analysis of a series of mutants of an Escherichia coli alanine transfer RNA shows that substitution of a single G-U base pair in the acceptor helix eliminates aminoacylation with alanine in vivo and in vitro. Introduction of that base pair into the analogous position of a cysteine and a phenylalanine transfer RNA confers upon each the ability to be aminoacylated with alanine. Thus, as little as a single base pair can direct an amino acid to a specific transfer RNA.

The tmRNA system performs translational surveillance and ribosome rescue in all eubacteria and some eukaryotic organelles. This system intervenes when ribosomes read to the 3' end of an mRNA or pause at internal codons with subsequent mRNA cleavage. A complex of alanyl-tmRNA (which functions as a tRNA and mRNA), SmpB protein, and EF-TucGTP binds stalled ribosomes, the nascent polypeptide is transferred to the alanine on tmRNA, and translation switches from the original message to a short tmRNA open reading frame (ORF) that encodes a degradation tag. Translation of the ORF and normal termination releases the tagged polypeptide for degradation and permits disassembly and recycling of ribosomal subunits for new rounds of protein synthesis. Structural and biochemical studies suggest mechanisms that keep tmRNA from interrupting normal translation and target ribosomes stalled with very short 3' mRNA extensions. Additional biological roles of tmRNA include stress management and the regulation of transcriptional circuits.