The SmpB C-terminal tail helps tmRNA to recognize and enter stalled ribosomes

Abstract

In bacteria, transfer-messenger RNA (tmRNA) and SmpB comprise the most common and effective system for rescuing stalled ribosomes. Ribosomes stall on mRNA transcripts lacking stop codons and are rescued as the defective mRNA is swapped for the tmRNA template in a process known as trans-translation. The tmRNA-SmpB complex is recruited to the ribosome independent of a codon-anticodon interaction. Given that the ribosome uses robust discriminatory mechanisms to select against non-cognate tRNAs during canonical decoding, it has been hard to explain how this can happen. Recent structural and biochemical studies show that SmpB licenses tmRNA entry through its interactions with the decoding center and mRNA channel. In particular, the C-terminal tail of SmpB promotes both EFTu activation and accommodation of tmRNA, the former through interactions with 16S rRNA nucleotide G530 and the latter through interactions with the mRNA channel downstream of the A site. Here we present a detailed model of the earliest steps in trans-translation, and in light of these mechanistic considerations, revisit the question of how tmRNA preferentially reacts with stalled, non-translating ribosomes.

Keywords: EFTu; SmpB; decoding; ribosome stalling; tmRNA.

FIGURE 1

tmRNA and SmpB binding in the decoding center. (A) Crystal structure of the Thermus thermophilus tmRNA–SmpB complex bound to EFTu on the 70S ribosome, trapped by kirromycin in the pre-accommodation state. Only the tRNA-like domain of tmRNA is included. Rendered using the coordinates from PDB 4ABR and 4ABS (Neubauer et al., 2012). (B) SmpB engages 16S rRNA nucleotides A1492, A1493, and G530 in the decoding center. The C-terminal tail extends into the mRNA channel. (C) Conserved SmpB residues interact with G530 and nearby nucleotides. T. thermophilus Tyr126 corresponds to E. coli His136. (D) Logo of the C-terminal tail from 470 SmpB proteins (Andersen et al., 2006) with the E. coli sequence shown underneath.

FIGURE 2

Model for selectivity in the early steps in trans-translation. EFTu delivers the Ala-tmRNA–SmpB complex to the A site regardless of whether the ribosome sits on a truncated message (top) or an intact message (bottom). Stacking of His136 on G530 in the decoding center triggers GTP hydrolysis and release of tmRNA from EFTu. These steps are independent of mRNA length. Binding of the SmpB C-terminal tail within the mRNA channel promotes accommodation of tmRNA into the A site in order to participate in peptidyl transfer (top). Intact mRNAs bias the equilibrium of the mRNA channel toward a closed conformation that blocks binding of the SmpB tail, leading to rejection of the tmRNA–SmpB complex on actively translating ribosomes (bottom).