A functional interaction of SmpB with tmRNA for determination of the resuming point of trans-translation

Abstract

In trans-translation, transfer-messenger RNA (tmRNA), possessing a dual function as a tRNA and an mRNA, relieves a stalled translation on the ribosome with the help of SmpB. Here, we established an in vitro system using Escherichia coli translation and trans-translation factors to evaluate two steps of trans-translation, peptidyl transfer from peptidyl-tRNA to alanyl-tmRNA and translation of the resume codon on tmRNA. Using this system, the effects of several mutations upstream of the tag-encoding region on tmRNA were examined. These mutations affected translation of the resume codon rather than peptidyl transfer, and one of them, A84U/U85G, caused a shift of the resume codon by À1. We also found that U 85 is protected from chemical modification by SmpB. In the A84U/U85G mutant, the base of protection was shifted from 85 to 84. Another mutation, A86U, which caused a shift of the resume codon by +1, shifted the base of protection from 85 to 86. The protection at 85 was suppressed by a mutation in the tRNA-like domain critical to SmpB binding. These results suggest that SmpB serves to bridge two separate domains of tmRNA to determine the initial codon for tag-translation. A mutant SmpB with a truncation of the unstructured C-terminal tail failed to promote peptidyl transfer, although it still protected U 85 from chemical modification.

SmpB as the handyman of tmRNA during trans-translation

“…A wealth of biochemical and genetic data have suggested that interactions between SmpB and key nucleotides lying upstream of the resume codon are instrumental in setting the new frame. 21,[70][71][72][73][74] At this step, the position of SmpB has been mapped by the sites of cleavages induced by hydroxyl radical probing of Fe(II) tethered SmpB mutants. Contrary to the A-site position that protruded into the mRNA path toward the downstream tunnel, they localize almost exclusively around the region of the P-site canonical codon-anticodon interaction.…”

Structural aspects of trans‐translation

“…Indeed, recently, an alternative binding site for SmpB on the A79-A86 loop was suggested (32). The protection of some nucleotides in this region has also been seen for the SmpBtmRNA complex in solution (33). Moreover, a number of mutations in this region either inactivate or affect trans-translation (34)(35)(36)47).…”

“…Besides the binding to the tRNA-like domain (TLD) of tmRNA, SmpB can interact with the sequence upstream of the resume codon (32,33).…”

“…The A79-A86 loop has been shown to be essential for correct resume codon positioning (34,35). Moreover, a mutation at position 86 allows for peptidyl transfer, but not for further translocation (33). The model predicts that pK1 should also play an essential role in the formation of such an element, being a structural constraint that determines the position of the SmpB-containing element in the ribosome.…”

tmRNA on its way through the ribosome

“…23,24 Several studies have demonstrated that mutations of the five nucleotides preceding the resume codon-the -1 triplet and the U85 and A86 nucleotides-induced frame-shifting in the translation of the ORF, and reduced or even completely abolished tmRNA tagging activity. 21,[24][25][26][27][28] Moreover, it has been shown that certain mutations in SmpB restore tagging activity of tmRNA mutated in the A86 nucleotide. 29,30 These studies suggested that the interaction of the SmpB with the upstream nucleotides plays a critical role in the selection of the correct reading frame.…”