The highest affinity binding site of small protein B on transfer messenger RNA is outside the tRNA domain

Abstract

Eubacterial ribosomes stalled on defective mRNAs are released through a mechanism referred to as trans-translation, depending on the coordinated actions of small protein B (SmpB) and transfer messenger RNA (tmRNA). A series of tmRNA variants with deletions in each structural domain were produced. Their structures were monitored by enzymatic and chemical probes in vitro, in the presence and absence of SmpB. Dissociation constants between these RNAs and SmpB from Aquifex aeolicus were derived by surface plasmon resonance (SPR) combined with filter binding assays. Three independent experimental evidences, including filter binding assays, SPR, and concentration titrations of the RNA-protein reactivity changes toward structural probes, indicate that the binding site that has the highest affinity for the protein is located outside the tRNA domain, upstream of the internal tag. The minimal tmRNA fragment that contains this high affinity site for SmpB, and also contains another site of lower affinity, includes the tag reading frame and three downstream pseudoknots that form a ring structure in solution.

SmpB as the handyman of tmRNA during trans-translation

“…Interaction of free SmpB with ribosomes is salt sensitive in vivo and therefore could be dependent upon the low stringency conditions used in the purification buffers, while its high binding affinity to tmRNA is unquestionable. 21 To our point of view, because SmpB was found in vivo bound to the ribosome and because the intracellular ionic environment can vary, we cannot rule out that this original route for initiating trans-translation might be used, at least under specific cellular conditions. 18 In any case, SmpB acts as a cellular sentinel onto the stalled ribosomes pinpointing those to be rescued.…”

“…A wealth of biochemical and genetic data have suggested that interactions between SmpB and key nucleotides lying upstream of the resume codon are instrumental in setting the new frame. 21,[70][71][72][73][74] At this step, the position of SmpB has been mapped by the sites of cleavages induced by hydroxyl radical probing of Fe(II) tethered SmpB mutants. Contrary to the A-site position that protruded into the mRNA path toward the downstream tunnel, they localize almost exclusively around the region of the P-site canonical codon-anticodon interaction.…”

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)*

“…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). Furthermore, recent structural studies have suggested that SmpB specifically recognizes stalled ribosomes through interactions between its C-terminal tail and the ribosomal mRNA channel (28).…”

Structural aspects of trans‐translation

“…The only space where it is possible to place SmpB is the loop formed by a single-stranded RNA region (A79-G87) and pK1; in our model, SmpB interacts with one of these elements (A79-G87). Indeed, recently, an alternative binding site for SmpB on the A79-A86 loop was suggested (32). The protection of some nucleotides in this region has also been seen for the SmpBtmRNA complex in solution (33).…”

“…Besides the binding to the tRNA-like domain (TLD) of tmRNA, SmpB can interact with the sequence upstream of the resume codon (32,33).…”