A previously uncharacterized role for small protein B (SmpB) in transfer messenger RNA-mediated trans-translation

Abstract

SsrA is a versatile RNA molecule found in all bacteria that functions as both a tRNA and an mRNA. SsrA rescues ribosomes stalled on damaged mRNAs and directs the tagging and degradation of their aberrant protein products. Small protein B (SmpB) is required for all known activities of SsrA. The two known functions of SmpB are binding SsrA RNA and promoting stable association of the SmpB.SsrA complex with 70S ribosomes. Using mutational analysis and biochemical experiments, we have discovered a previously uncharacterized SmpB function. This function is required for a step in the tagging process downstream of SsrA binding and ribosome association but before transpeptidation of the SsrA-linked alanine and establishment of the SsrA reading frame. Our results clearly demonstrate that residues in the C-terminal tail of SmpB confer a hitherto unrevealed function that is essential for trans-translation. Based on these results, we propose that upon binding stalled ribosomes, the unstructured C-terminal tail of SmpB acquires contacts that are critical for productive accommodation of SsrA into the ribosomal A site.

Fig. 1.

Endogenous tagging phenotypes. (A) Western blot analysis using IR800-conjugated anti-his6 antibody, with the pattern of proteins tagged by SsrA^H6 in cells expressing different SmpB variants. (B) Analysis of λimmP22 hybrid phage induction supported by different SmpB mutants. Data are presented as efficiency of plating (EOP), where the number of plaques formed when SmpB^WT was expressed is taken as EOP = 1. We have used SmpB^WT as a positive control and SmpB⁵⁹ (an SmpB truncation mutant with only residues 1–59) as a negative control throughout these experiments.

Fig. 2.

SsrA-binding assays. (Upper) Gel mobility-shift assays of the SsrA-binding propensity of SmpB^WT and SmpB¹³⁹. (Lower) Curve-fit analysis used to determine the apparent equilibrium dissociation constants (K_d) of SsrA¹¹³–SmpB interactions.

Fig. 3.

Ribosome association. (A and B) Northern blot analysis using an SsrA-specific probe to detect SsrA RNA in purified ribosome preparations. (C and D) Ethidium bromide staining of the same gel as in A and B, shown to demonstrate that similar amounts of ribosomal RNA were loaded in each lane. (E and F) Western blot analysis using anti-his6 antibody to detect his6-tagged SmpB protein in the same purified ribosome preparations used in A–D. The SmpB variant expressed in the cells from which the ribosomes were purified is indicated on the horizontal axis.

Fig. 4.

MALDI-TOF MS spectra of purified λ-N protein from cells expressing the SmpB^AA, SmpB^DE, SmpB¹⁵³, and SmpB¹⁴⁸ variants. The species with m/z = 13,102 is the major untagged λ-N protein product, whereas the peak with m/z = 14,427 corresponds to the major product with the full SsrA^H6 encoded tag sequence. For details of the experiment and results, see Fig. 6.

Fig. 5.

Phenotypes of mutations to the 137–139 region. (A) Western analysis of endogenous tagging activity. (B) Bar graph depicting the mean and standard deviation of the percent of wild-type endogenous tagging signals for three separate experiments. (C) Northern blot using an SsrA-specific probe showing copurification of SsrA RNA with ribosomes. (D) Western blot showing the presence of SmpB in the same ribosome preparation as in C. The samples in C and D were normalized by A₂₆₀ to ensure that a similar amount of ribosomes was loaded into each lane (data not shown).