Protein factors associated with the SsrA.SmpB tagging and ribosome rescue complex

Abstract

SsrA RNA acts as a tRNA and mRNA to modify proteins whose synthesis on ribosomes has stalled. Such proteins are marked for degradation by addition of peptide tags to their C termini in a reaction mediated by SsrA RNA and SmpB, a specific SsrA-RNA binding protein. Evidence is presented here for the existence of a larger ribonucleoprotein complex that contains ribosomal protein S1, phosphoribosyl pyrophosphate synthase, RNase R, and YfbG in addition to SsrA RNA and SmpB. Biochemical, genetic, and phylogenetic results suggest potential roles for some of these factors in various stages of the ribosome rescue and tagging process and/or the presence of functional interactions between one or more of these proteins and SsrA.

Figure 1

Predicted secondary structure of SsrA RNA (4). The tRNA-like domain, pseudoknots, and degradation tag-coding sequence are marked.

Figure 2

Purification of SsrA⋅SmpB associated factors. (A) Elution of complex from Sephacryl S300 gel-filtration column. The elution profile of SmpB protein alone is shown for comparison. (Inset) Western and Northern blot analysis of the main peak for the presence SmpB protein and SsrA RNA, respectively. (B) SDS/PAGE analysis following Ni²⁺-NTA and Sephacryl S300 columns. After each purification step, samples were concentrated 10-fold and electrophoresed on a 12% Laemmli SDS gel. Arrows mark the positions of identified polypeptides. Molecular mass markers are shown on right.

Figure 3

(A) Gel-mobility shift assay of the binding of purified His₆-tagged ribosomal protein S1 to 100 pM of ³²P-labeled SsrA RNA produced by transcription in vitro. Arrows mark the positions of the bound and free RNA species. (B) Binding curve generated from data in A. (C) Binding of His₆-tagged ribosomal protein S1 to 100 pM ³²P-labeled PK1L and PK3L SsrA variants.

Figure 4

Gel-mobility shift assay of the binding of purified His₆-tagged PrsA protein to 100 pM of ³²P-labeled SsrA RNA. Arrows mark the bound and free positions.

Figure 5

Western-blot analysis of SsrA–H₆-tagged proteins in W3110/pKW24 cells, W3110 smpBΔ-1/pKW24 cells, and W3110 rnr∷cm/pKW24 cells. Cell lysates from each strain were prepared as described in Materials and Methods, resolved by electrophoresis on a 12% SDS/PAGE, transferred to PVDF membrane, and probed with anti-His₆ antiserum.