Figure 1.
Schematic depiction of protein S1 and its variants used in this study.
All protein variants were C-terminally FLAG-tagged to facilitate detection. For pull-down assays shown in Figure 4 and for protein purification, protein variants contained an additional N-terminal HIS-tag.
Figure 2.
Protein S1106 affects E. coli growth by displacing native S1 from the ribosome.
(A) In contrast to synthesis of protein S187–194 (representing domain D2, -•-), synthesis of S1106 and S1194 (representing domains D1 (-▪-) and D1-2 (-▴-)) inhibits bacterial growth. E. coli strain JE28 harbouring plasmids pProEX-HTb (-◊-), pPro-S1D1F (-▪-), pPro-S1D2F (-•-) and pPro-S1D1-2F (-▴-) were grown in LB medium containing ampicilin (100 µg/ml) and kanamycin (20 µg/ml). At OD600 of 0.2–0.25 (indicated by an arrow) 50 µM IPTG was added to the cultures. Aliquots were withdrawn from each culture for ribosome preparation 1 hour upon induction. (B) Proteins present in S30 extracts (lanes 1, 3, 5, and 7) and 70 S ribosomes (lanes 2, 4, 6, and 8) prepared from cells without overexpression (lanes 1 and 2), and cells overexpressing S1106 (lanes 3 and 4), S187–194 (lanes 5 and 6), or S1194 (Lanes 7 and 8) were separated on a 12.5% SDS-PAGE and presence of protein S1 and its variants on 70 S ribosomes was checked by western blot analysis using anti-S1 antibodies (panel a), anti-FLAG (panel b) and anti-L2 antibodies (panel c), which served as loading control. The positions of the respective proteins are indicated to the right. (C) The N-terminal domain of S1 is required for assembly to the ribosome. Equimolar amounts of HIS-tagged ribosomes (lanes 1 and 3) and ribosome free S100 extract (lanes 2 and 4) purified from E. coli strain JE28 overexpressing FLAG-tagged proteins S1 (lanes 1 and 2) and S187–557, lacking domain D1 (lanes 3 and 4) were separated on a 12.5% SDS-PAGE. The presence of S1 and S187–557 was determined by western blot analysis employing anti-FLAG antibodies (panel a) and anti-L2 antibodies (panel b), which served as loading control.
Table 1.
Bacterial strains and plasmids used in this study.
Figure 3.
Domain D2 is not involved in ribosome binding of protein S1.
(A) Ribosome binding of proteins S1 or S1Δ103–181 was determined 60 minutes upon induction of their synthesis in strain JE28 harbouring either plasmid pPro-S1F (lanes 1 and 2) or pPro-S1ΔD2F (lanes 3 and 4). S30 extracts (lanes 1 and 3) and purified 70 S ribosomes (lanes 2 and 4) were loaded on SDS-PAGE. The positions of proteins S1 and S1Δ103–181 are indicated to the right. (B) The binding of S1 (lanes 5–7) or S1Δ103–181 (lanes 8–10) for the ribosome was determined by in vitro reconstitution experiments employing 30 S(-S1) subunits. The affinity of both proteins was directly compared by a competition experiment incubating 30 S (-S1) ribosomes concomitantly with both proteins S1 and S1Δ103–181 in equimolar amounts (lanes 12–14). Upon incubation the ribosomes were separated from unbound proteins as described in Material and Methods, and the proteins present in the different fractions were separated on SDS-PAGE and visualized by Coomassie staining. I, input (lanes 2, 5, 8, and 12); R, ribosome fractions (lanes 3, 6, 9, and 13); FT, flow through fractions (lanes 4, 7, 10, and 14). 30 S, 30 S ribosomes before depletion for protein S1 (lane 15); 30 S(-S1), S1 depleted ribosomes used for the study (lane 16). The positions of proteins S1 and S1Δ103–181 are indicated to the right. Lanes 1 and 11, protein size marker.
Figure 4.
Synthesis of S1 variants S1106 and S1194 results in selective translation of lmRNAs.
Pulse labelling of strain JE28 harbouring plasmids encoding proteins S1106 (lanes 1–4), S187–194 (lanes 5–8), and S1194 (lanes 9–12) was carried out before (time point 0) and 15, 30, and 60 min upon induction as described in Materials and Methods. Labelled proteins were separated on a 12.5% SDS-PAGE. Positions of proteins S1106, S187–194 and S1194 (marked by asterisks) and the position of the CI-LacZ fusion protein encoded by a lmRNA are indicated to the right of the autoradiograph.
Figure 5.
S1 variants S1106 and S1194 directly interact with protein S2.
S30 extracts (Input, I; lanes 1, 3, 5, and 7) prepared from E. coli strain Tuner harbouring the empty vector pProEX-HTb (lanes 1 and 2) and its derivatives encoding proteins S1106 (lanes 3 and 4), S187–194 (lanes 5 and 6) and S1194 (lanes 7 and 8) were loaded onto Ni-NTA agarose to allow binding of the HIS-tagged S1 variants. After washing with 10 column volumes the proteins bound to the matrix were eluted (Elution, E; lanes 2, 4, 6, and 8). The presence of protein S1 variants in input and elution fractions was checked employing anti-FLAG antibodies (panel a). Likewise, both fractions were assayed for the co-purification of protein S2 by SDS-PAGE followed by western blot analysis employing an anti-S2 antibody (panel b).
Figure 6.
Protein S1 interacts with the coiled-coil domain of S2 via its N-terminal domain.
(A) Position of protein S2 on the 30 S subunit. The structure of the 30 S subunit was modelled employing Polyview 3D [46] and PyMOL molecular system software [47] and PDB file 2AVY [48]. The 16 S rRNA and proteins are shown in light and dark grey, respectively. The globular domain and the coiled-coil domain of S2 are indicated in green and red, respectively. (B) Enlargement and clockwise rotation of the structure shown in (A). The coiled-coil domain of protein S2 (S2α2; red) interacts with helices h35–h37 (blue) within the head of the 30 S subunit, whereas the globular domain (green) contacts helix h26 (magenta) in the body of the 30 S subunit. The tentative position of domain D1 of protein S1 interacting with the S2α2 domain is indicated by a blue sphere. (C) S30 extracts containing either FLAG-tagged proteins S1, S187–557, or the HIS-tagged protein S2α2 were mixed. An aliquot was subjected to western blot analysis to determine the amount of respective proteins present (lanes 1 and 2). Then the mixture was applied to a Ni-NTA column. Upon washing protein S2α2 was eluted and the co-purification of S1 (lane 3) or S187–557 (lane 4) was checked by western blotting using anti-FLAG antibodies (panel b). The amount of protein S2α2 was determined using anti-S2 antibodies (panel a). (D) 70 S ribosomes were purified from E. coli strain Tuner (DE3) before (lane 1; time point 0) and 30, 60 and 90 min upon induction of protein S2α2 synthesis (lanes 2–4). The presence of proteins S1, S2, and S2α2 was determined by western blotting using anti-S1 (panel a) and anti-S2 antibodies (panels b and c), respectively. The amount of protein S10, which served as an internal control was determined by anti-S10 antibodies (panel c). Lane 5, purified S2α2 was loaded to unambiguously identify the protein.
Table 2.
Synthetic oligonucleotides used in this study.