Function of the SmpB Tail in Transfer-messenger RNA Translation Revealed by a Nucleus-encoded Form

Abstract

Stalled bacterial ribosomes are freed when they switch to the translation of transfer-messenger RNA (tmRNA). This process requires the tmRNA-binding and ribosome-binding cofactor SmpB, a ␤-barrel protein with a protruding C-terminal tail of unresolved structure. Some plastid genomes encode tmRNA, but smpB genes have only been reported from bacteria. Here we identify smpB in the nuclear genomes of both a diatom and a red alga encoding a signal for import into the plastid, where mature SmpB could activate tmRNA. Diatom SmpB was active for tmRNA translation with bacterial components in vivo and in vitro, although less so than Escherichia coli SmpB. The tail-truncated diatom SmpB, the hypothetical product of a misspliced mRNA, was inactive in vivo. Tail-truncated E. coli SmpB was likewise inactive for tmRNA translation but was still able to bind ribosomes, and its affinity for tmRNA was only slightly diminished. This work suggests that SmpB is a universal cofactor of tmRNA. It also reveals a tail-dependent role for SmpB in tmRNA translation that supersedes a simple role of linking tmRNA to the ribosome, which the SmpB body alone could provide.

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

“…Thus, the C-terminal tail may have a role in the interaction with the ribosome rather than tmRNA during trans-translation. This is consistent with a previous finding that a truncation of the C-terminal tail only slightly reduces the affinity with tmRNA (Jacob et al 2005). The site of this interaction on SmpB has yet to be determined.…”

“…In a crystal structure of SmpB in complex with a fragment of the tRNA-like domain (TLD) (Gutmann et al 2003), SmpB binds the elbow of TLD, in agreement with results of biochemical studies (Hanawa-Suetsugu et al 2002;Nameki et al 2005), and SmpB can be superimposed on the anticodon stem and loop of tRNA, if TLD is fixed on the amino acid acceptor stem and the T-arm, implying that SmpB functions around the decoding region during trans-translation. Although with no structural information about the C-terminal region, it has recently been shown that SmpB having a mutation or truncation in the C-terminal tail does not facilitate trans-translation in vivo (Jacob et al 2005;Sundermeier et al 2005). This raises the possibility that the C-terminal tail introduces the first GCA codon of tmRNA to the A-site of the 70S ribosome by interacting with some part of tmRNA such as the upstream sequence in proximity to the decoding center of the ribosome.…”

“…No involvement of the C-terminal tail of SmpB in the interaction with the upstream region of the tag-encoding sequence of tmRNA It has been demonstrated that the C-terminal tail of SmpB is required for trans-translation in vivo but not for binding to tmRNA and promoting its association with the ribosome (Jacob et al 2005;Sundermeier et al 2005). We prepared a mutant SmpB with a truncation of C-terminal residues from 133 to 160 (D133 to 160).…”

The role of SmpB and the ribosomal decoding center in licensing tmRNA entry into stalled ribosomes

“…2). While the helical propensity of the SmpB tail has been noted for some time (Jacob et al 2005), it has never been determined whether helix formation plays FIGURE 2. Alignment of the SmpB C-terminal tail.…”

SmpB as the handyman of tmRNA during trans-translation

“…First of all it does not contribute to the binding of the protein to the ribosome, nor to the GTP hydrolysis by EF-Tu as demonstrated by testing various mutated or tail-truncated proteins. 10,39,48,49 Therefore, its function is crucial for the events that are after the initial association with the ribosome but before transpeptidation. 39 It is suggested that The density attributable to 'tmrNA-SmpB' is in red, the 50S subunit is blue, the 30S subunit is yellow, and the P-site and e-site trNAs are depicted in green and orange, respectively, and the problematic mrNA is purple.…”