Resolving nonstop translation complexes is a matter of life or death

Abstract

Problems during gene expression can result in a ribosome that has translated to the 3' end of an mRNA without terminating at a stop codon, forming a nonstop translation complex. The nonstop translation complex contains a ribosome with the mRNA and peptidyl-tRNA engaged, but because there is no codon in the A site, the ribosome cannot elongate or terminate the nascent chain. Recent work has illuminated the importance of resolving these nonstop complexes in bacteria. Transfer-messenger RNA (tmRNA)-SmpB specifically recognizes and resolves nonstop translation complexes in a reaction known as trans-translation. trans-Translation releases the ribosome and promotes degradation of the incomplete nascent polypeptide and problematic mRNA. tmRNA and SmpB have been found in all bacteria and are essential in some species. However, other bacteria can live without trans-translation because they have one of the alternative release factors, ArfA or ArfB. ArfA recruits RF2 to nonstop translation complexes to promote hydrolysis of the peptidyl-tRNAs. ArfB recognizes nonstop translation complexes in a manner similar to tmRNA-SmpB recognition and directly hydrolyzes the peptidyl-tRNAs to release the stalled ribosomes. Genetic studies indicate that most or all species require at least one mechanism to resolve nonstop translation complexes. Consistent with such a requirement, small molecules that inhibit resolution of nonstop translation complexes have broad-spectrum antibacterial activity. These results suggest that resolving nonstop translation complexes is a matter of life or death for bacteria.

FIG 1

Mechanisms for resolving nonstop translation complexes. During trans-translation (top), tmRNA-SmpB recognizes nonstop translation complexes by binding in the empty mRNA channel and uses a reading frame within tmRNA to mediate the release of the ribosome and target the nascent polypeptide for proteolysis. The problematic mRNA is also degraded. Some bacteria have backup systems that use either ArfA or ArfB to recognize nonstop translation complexes. ArfA recruits RF2, which uses its GGQ motif to hydrolyze the peptidyl-tRNA in the ribosome. It is not known how ArfA recognizes nonstop translation complexes, but it might bind in the empty mRNA channel in a manner similar to that of SmpB and ArfB binding. ArfB contains a GGQ motif and directly hydrolyzes the peptidyl-tRNA on the ribosome. ArfA and ArfB release the ribosome but do not target the nascent polypeptide for degradation. See the text for details.

FIG 2

Recognition of nonstop translation complexes. Structure models of an elongation complex (A) with an intact mRNA compared to recognition of nonstop translation complexes by tmRNA-SmpB (B) and ArfB (C) are shown. The 30S ribosomal subunits are shown in gray, with decoding nucleotides G530, A1492, and A1493 in white. (A) An elongation complex trapped by kirromycin from PDB 2WRQ, with mRNA (purple), E-site tRNA (yellow), P-site tRNA (blue), and A-site tRNA (green) bound with EF-Tu (orange). (B) trans-Translation complex trapped by kirromycin from PDB 4ABR. The tRNA-like domain of tmRNA (pink) bound with EF-Tu (orange) is in an orientation similar to that of the acceptor stem of the tRNA shown in panel A. SmpB (green) occupies the codon-anticodon region and extends into the empty mRNA channel. (C) In nonstop translation complexes recognized by ArfB (from PDB 4DH9), ArfB (green) extends into the empty mRNA channel, with the catalytic GGQ domain near the peptidyl-tRNA in the P site (blue).

FIG 3

Phylogenetic distribution of trans-translation, ArfA, and ArfB. Species in which the phenotype of deleting ssrA or smpB is known are shown on a phylogenetic tree based on 16S rRNA sequences. Bold names indicate species in which ssrA or smpB is essential. The presence of genes encoding tmRNA-SmpB, ArfA, and ArfB is shown. For ArfA and ArfB, a filled box indicates that the system is sufficient to maintain viability in the absence of tmRNA-SmpB, an empty box indicates that the system is not sufficient to maintain viability in the absence of tmRNA-SmpB, and a hashed box indicates that it is not yet known whether the system is sufficient to maintain viability. Salmonella typhimurium, Salmonella enterica serovar Typhimurium.

FIG 4

High-throughput screening assay to identify trans-translation inhibitors. The reporter contains a gene encoding luciferase with a strong transcriptional terminator inserted before the stop codon, such that transcription results in a nonstop mRNA. E. coli cells containing the reporter were screened in high-throughput format to identify compounds that inhibit trans-translation. When no inhibitor is present, translation of the nonstop mRNA results in trans-translation followed by proteolysis of luciferase, and cells produce no luminescence. Conversely, active luciferase is produced when a trans-translation inhibitor is present, resulting in luminescence.