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

Abstract

In bacteria, stalled ribosomes are recycled by a hybrid transfer-messenger RNA (tmRNA). Like tRNA, tmRNA is aminoacylated with alanine and is delivered to the ribosome by EF-Tu, where it reacts with the growing polypeptide chain. tmRNA entry into stalled ribosomes poses a challenge to our understanding of ribosome function because it occurs in the absence of a codonanticodon interaction. Instead, tmRNA entry is licensed by the binding of its protein partner, SmpB, to the ribosomal decoding center. We analyzed a series of SmpB mutants and found that its C-terminal tail is essential for tmRNA accommodation but not for EF-Tu activation. We obtained evidence that the tail likely functions as a helix on the ribosome to promote accommodation and identified key residues in the tail essential for this step. In addition, our mutational analysis points to a role for the conserved K 131 GKK tail residues in trans-translation after peptidyl transfer to tmRNA, presumably EF-G-mediated translocation or translation of the tmRNA template. Surprisingly, analysis of A1492, A1493, and G530 mutants reveals that while these ribosomal nucleotides are essential for normal tRNA selection, they play little to no role in peptidyl transfer to tmRNA. These studies clarify how SmpB interacts with the ribosomal decoding center to license tmRNA entry into stalled ribosomes.

Active and Accurate trans-Translation Requires Distinct Determinants in the C-terminal Tail of SmpB Protein and the mRNA-like Domain of Transfer Messenger RNA (tmRNA)*

“…We found that alanine substitution at this position, or substitution of conserved lysines in the region encompassing residues 131-134, decreased the tagging ability of SmpB. This finding is qualitatively consistent with previous studies (30), although the defects we observed were not as severe. Nonetheless, the SmpB G132D and SmpB K131A/K133A/K134A substitutions demonstrate that this region plays a critical role in SmpB function during trans-translation.…”

“…Several conserved nucleotides are essential constituents of this region, alterations of which have severe consequences on tmRNA tagging activity. For instance, substitution of the adenine at position 86 (A86) with cytosine in the E. coli tmRNA nearly abolishes all tagging activity (30,40). This particular sub- .…”

“…SmpB binds specifically and with high affinity to the tRNA-like domain of tmRNA and is necessary for the recruitment and stable association of the SmpB-tmRNA complex with stalled ribosomes (15,30). SmpB is also required for a novel function following ribosome association but prior to the addition of the tmRNA alanine charge (27).…”

“…SmpB variants that lack the C-terminal tail support delivery of tmRNA to stalled ribosomes but are unable to assist in subsequent steps of trans-translation (27). A number of genetic and structural studies have suggested that the C-terminal tail of SmpB is necessary for accommodation and transpeptidation and ultimately the establishment and selection of the tmRNA ORF (27,30,31). This task might be accomplished by direct interactions between the C-terminal tail of SmpB and the mRNA-like domain of tmRNA (32)(33)(34).…”

Ribosomal Protein S1: An Important Trans-Translational Factor

“…Within the TLD-SmpB complex, SmpB mimics the anticodon arm of a canonical tRNA [6]. The C-terminal tail of SmpB has been shown to mimic mRNA, and play an essential role in the functioning of ribosome-bound tmRNA [7,8].…”