Pre-binding of Small Protein B to a Stalled Ribosome Triggers trans-Translation

Abstract

To rescue stalled ribosomes, eubacteria employ a molecule, transfer messenger RNA (tmRNA), which functions both as a tRNA and as an mRNA. With the help of small protein B (SmpB), tmRNA restarts protein synthesis and adds by the trans-translation mechanism a peptide tag to the stalled protein to target it for destruction by cellular proteases. Here, the cellular location and expression of endogenous SmpB were monitored in vivo. We report that SmpB is associated with 70S ribosomes and not in the soluble fraction, independently of the presence of tmRNA. In vitro, SmpB that is pre-bound to a stalled ribosome can trigger initiation of trans-translation. Our results demonstrate the existence of a novel pathway for the entry of tmRNA to the ribosome and for the trans-transfer of a nascent peptide chain from peptidyl-tRNA to charged tmRNA.

Stepping Transfer Messenger RNA through the Ribosome

“…According to cryo-EM data, SmpB is bound to tmRNA in the pre-accommodation complex (17). Recently, it was found that SmpB can also be bound to the ribosome (16) and that two molecules of SmpB pre-bound to one ribosome facilitate the accommodation of tmRNA in an in vitro system. These data indicate that SmpB plays an essential role at the stage of the initiation of trans-translation.…”

“…Recently, it was shown that all SmpB molecules are bound to ribosomes within the cell. Moreover, one ribosome can bind two protein molecules that remain bound to the ribosome even after the transfer of undersynthesized peptide to tmRNA (16). SmpB was also found to be the partner of tmRNA on entering the ribosome during the pre-accommodation stage (17).…”

SmpB as the handyman of tmRNA during trans-translation

“…The SmpB protein is an essential component for trans-translation in vivo, responsible of a variety of actions in the course of the mechanism. It binds with high affinity to tmRNA in vivo, 9 protects it from degradations in the cells (3-fold shorter half-life of tmRNA in the Δsmpb cells compared with wild-type cells, Hallier et al 18 ) and enhances its alanylation efficiency.…”

“…While SmpB is required for ala-tmRNA to bind the stalled ribosomes, the opposite is not true: SmpB can bind tightly to 70S ribosomes in the absence of tmRNA, either in vitro or in vivo. 18,19 This raises the question of the chronology of the early events leading to trans-translation initiation. Is a pre-formed Figure 1.…”

Lost in translation

“…Lorsque des ribosomes en cours de traduction parviennent à l'extrémité d'ARN messagers défectueux, une petite protéine basique appelée petite protéine B ou SmpB [11], possédant certaines analogies avec des protéines du ribosome, s'associe avec les ribosomes bloqués [12,13]. Ce positionnement prépare l'arrivée de l'ARNtm.…”