Abstract
Unproductive ribosome stalling in eubacteria is resolved by the actions of SmpB protein and transfer messenger (tm) RNA. We examined the functional significance of conserved regions of SmpB and tmRNA to the trans-translation process. Our investigations reveal that the N-terminal 20 residues of SmpB, which are located near the ribosomal decoding center, are dispensable for all known SmpB activities. In contrast, a set of conserved residues that reside at the junction between the tmRNA-binding core and the C-terminal tail of SmpB play an important role in tmRNA accommodation. Our data suggest that the highly conserved glycine 132 acts as a flexible hinge that enables movement of the C-terminal tail, thus permitting proper positioning and establishment of the tmRNA open reading frame (ORF) as the surrogate template. To gain further insights into the function of the SmpB C-terminal tail, we examined the tagging activity of hybrid variants of tmRNA and the SmpB protein, in which the tmRNA ORF or the SmpB C-terminal tail was substituted with the equivalent but highly divergent sequences from Francisella tularensis. We observed that the hybrid tmRNA was active but resulted in less accurate selection of the resume codon. Cognate hybrid SmpB was necessary to restore activity. Furthermore, accurate tagging was observed when the identity of the resume codon was reverted from GGC to GCA. Taken together, these data suggest that the engagement of the tmRNA ORF and the selection of the correct translation resumption point are distinct activities that are influenced by independent tmRNA and SmpB determinants.
Highlights
TmRNA and small protein B (SmpB) rescue stalled ribosomes through a template switching mechanism
To gain further insights into the function of the SmpB C-terminal tail, we examined the tagging activity of hybrid variants of tmRNA and the SmpB protein, in which the tmRNA open reading frame (ORF) or the SmpB C-terminal tail was substituted with the equivalent but highly divergent sequences from Francisella tularensis
SmpB Glycine 132 Is Important for trans-Translation—Previous studies have established that the SmpB protein is a requisite factor in the tmRNA-mediated ribosome rescue system [7, 9, 10]
Summary
SmpBGly-132 Mutagenesis and Screening—To generate a pool of SmpB variants at amino acid position 132 of SmpB, plasmid pET28BAH6 containing Escherichia coli SmpB and a tmRNA variant encoding a tag containing six histidines, tmRNAH6, were mutagenized using the QuikChange mutagenesis kit with the following primers: Gly-132-random reverse (5Ј-GTTTATCGTGCTGTTTCTTNNNTTTGGCGACGCCG-3Ј) and Gly-132-random forward (5Ј-CGGCGTCGCCAAANNNAAGAAACAGCACGATAAAC-3Ј), where NNN is the randomized Gly-132 codon. -Nonstop Reporter Expression—E. coli strain W3110 ⌬smpBssrA (DE3), which lacks the necessary components for trans-translation, was transformed with pKW846 This plasmid contains a T7-driven truncated variant of the N-terminal. Domain of the cI repressor (hereafter -cI), followed by the trpA gene transcriptional terminator (trpAt) This strain was transformed with an additional plasmid, encoding the indicated variants of SmpB and tmRNA under the control of their native promoter. Tight-coupled 70 S ribosomes were resuspended in 500 l of resuspension buffer (20 mM Tris, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 0.5 mM EDTA, 6 mM -mercaptoethanol, 10 units/ml SuperASE In) and subjected to Western and Northern blot analyses. Cells were harvested and resuspended in lysis buffer (1 M NH4Cl, 150 mM KCl, 50 mM Tris, pH 8.0, 2 mM -mercaptoethanol, 20 mM imidazole) and lysed by sonication. The membrane-bound proteins were probed with primary antibodies to the His or the FLAG epitope and secondary antibody conjugated to a fluorescent dye, detected using the Odyssey infrared imaging system, and quantified using the Odyssey data analysis software (LI-COR and ImageJ)
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