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

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 -1. 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.