Making the jump: new insights into the mechanism of trans-translation
© BioMed Central Ltd 2008
Published: 30 June 2008
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© BioMed Central Ltd 2008
Published: 30 June 2008
The transfer-messenger ribonucleoprotein (tmRNP), which is composed of RNA and a small protein, small protein B (SmpB), recycles ribosomes that are stalled on broken mRNAs lacking stop codons and tags the partially translated proteins for degradation. Although it is not yet understood how the ribosome gets from the 3' end of the truncated message onto the messenger portion of the tmRNA to add the tag, a recent study in BMC Biology has shed some light on this astonishing feat.
The mechanism of trans-translation however is mysterious. Because the TLD of tmRNA has no anticodon, it is not clear how it can recognize and bind to the empty A site of a stalled ribosome (Figure 2). Moreover, the MLD has neither an AUG start codon nor the Shine-Dalgarno sequence whereby bacterial mRNA binds to a complementary region of the ribosomal RNA at the start of translation. How then is the resume triplet properly positioned? And what mechanism allows the ribosome to take off from the damaged mRNA template and land precisely on the tmRNA's resume codon? Astonishingly, the ribosome performs this feat when a peptide bond forms between the partially synthesized protein and the alanine-charged tmRNA, and while establishing the correct reading frame for continuing elongation. Miller and colleagues  have now carried out a systematic site-directed mutagenesis study in an attempt to establish the contribution of the nucleotide residues that precede the resume codon to the correct positioning of the MLD.
One problem in determining the critical elements of trans-translation in vivo has been that E. coli cells grow well without the ssrA gene, so mutations cannot be detected by their effects on growth. Furthermore, the tagged proteins produced by trans-translation are degraded, and therefore cannot be used to indicate whether it is occurring normally. Luckily, however, a wide variety of tag templates are tolerated, and, upon removal of the natural stop codons, large additions can be engineered onto the tmRNA and are then translated . The group of Allen Buskirk has used an ingenious assay in which proper tagging of truncated kanamycin resistance (KanR) gene products on stalled ribosomes produces full-length KanR protein, so that E. coli survives on kanamycin plates only when the tmRNP is functional .
The new systematic in vivo study from the Buskirk laboratory that has recently been published in BMC Biology  provides strong experimental evidence that the previously suspected -1 resume triplet has only a minor role in accommodating tmRNA on the ribosome. In this paper, Miller and colleagues  constructed mutant tmRNAs with all 64 possible permutations of the -1 triplet and determined their effect on survival in the kanamycin resistance assay. They found that eight of the 18 codons that were prohibited according to the -1 hypothesis  were in fact fully functional, and other mutant tmRNAs that were predicted by the -1 triplet rule to be functional were shown by experiment to be completely inactive. The results of this comprehensive study show that the proposed rule for the -1 triplet is invalid and suggest different nucleotides that are important for accommodation of tmRNA on the ribosome.
One alternative nucleotide is the highly conserved adenosine at position 86 of E. coli tmRNA (Figure 3), which was observed earlier to be important in trans-translation . Indeed, by measuring survival in the kanamycin-resistance assay, the investigators confirmed that changing A86 to a pyrimidine yielded cells that were unable to trans-translate.
Because high-resolution structures of the ribosome-bound tmRNA at various stages of trans-translation are currently unavailable, it is unclear why the conserved A86 has such a prominent role. Although this adenosine residue may act independently to interact with the ribosome, the investigators suggest that the A86 interacts with a yet to be identified ligand that is primarily responsible for engaging the resume triplet and tmRNA in the attachment and synthesis of the tag peptide. They speculate that A86 might bind to the SmpB that is part of the transfer-messenger RNA ribonucleoprotein, or to ribosomal protein S1, two proteins that have been found by other investigators to be close to the decoding center of the ribosome-bound tmRNA at some stage of trans-translation [14–18]. Further studies at the atomic level will be required before the athletic potential of the ribosome is fully understood.
The authors were supported by grants GM58267 and GM49034 from the NIH. We dedicate this work to the late Twix.