Overexpression of RimJ alters ribosome profiles of the S5(G28D) strain. Sucrose gradient sedimentation analysis of ribosomes isolated from the S5(G28D) mutant strains with empty pBAD (pBAD) and RimJ overexpressed (p-rimJ). Positions of the 30S and 50S subunits and 70S ribosomes are indicated. Ribosome analysis of S5(G28D) strain with pBAD grown at 37°C (A), p-rimJ grown at 37°C (B), pBAD grown at 20°C (C), p-rimJ grown at 20°C (D). Ribosome analyses of the parental CSH142 strain grown at 37°C (E) or 20°C (F) are shown for comparison.

RimJ can suppress defects associated with S5(G28D) independent of its acetyltransferase activity. Overexpression of wild-type RimJ in the ΔrimJ or S5(G28D) background by induction of p-rimJ. Mutant RimJ proteins were overexpressed from p-rimJ(C105A), p-rimJ(C54A, C105A) and p-rimJ(C54F, C105A). Numbers and letters after the cysteines (C) correspond to positions in E. coli RimJ sequence that have been mutated to alanine (A) or phenylalanine (F). A. Multiple alignment of RimJ sequences and positions of C54 and C105. Sequence comparisons of RimJ from various prokaryotic species. The NAT domain in E. coli RimJ sequence is underlined. C54 and C105 residues are boxed. The RimJ sequences used in sequence alignment and their accession numbers in parenthesis are as follows. Escherichia: Escherichia coli (NP_415584), Shigella: Shigella flexneri (P0A950), Salmonella: Salmonella enterica (NP_455660), Yersinia: Yersinia pestis (CAL20676), Vibrio: Vibrio cholerae (YP_001216873), Pseudomonas: Pseudomonas aeruginosa (YP_791309). B. Western analysis for acetylated proteins in 30S subunit proteins (TP30) obtained from the ΔrimJ strain (CWZ387) with overexpression of various constructs: (1) p-rimJ (2) p-rimJ(C105A) (3) p-rimJ(C54A, C105A) (4) p-rimJ(C54F, C105A). C. 10-fold serial dilutions of S5(G28D) with various constructs at the non-permissive temperature, 20°C: (1) p-rimJ(C54A, C105A) (2) p-rimJ (3) empty pBAD. D. Sucrose gradient sedimentation analysis of ribosomes from S5(G28D) grown at the non-permissive temperature, 20°C, with the constructs: (1) p-rimJ(C54A, C105A) (2) p-rimJ (3) empty pBAD. Positions of the 30S and 50S subunits and 70S ribosomes are indicated.

Analysis of growth of S5(G28D) strain in the presence and absence of RimJ. A and B. Growth of S5(G28D) cells in the absence or presence of RimJ induction as shown by the 10-fold serial dilutions of S5(G28D) with pBAD vector (empty pBAD) or with the same vector containing the rimJ gene for overexpression (p-rimJ) at the permissive, 37°C (A) and non-permissive temperature, 20°C (B). Plates contain arabinose to allow continued expression of rimJ. C. Wild-type parental (BW25113), ΔrimJ (JW1053), S5(G28D) and the double mutant [S5(G28D) ΔrimJ] are grown at the permissive, 37°C on LB plates. Corresponding doubling times in minutes for growth in liquid LB media (with appropriate antibiotics or arabinose) are also indicated for each strain at the bottom.

RimJ rescues translational miscoding of S5(G28D). Nonsense suppression and frameshifting as monitored by β-galactosidase activity in S5(G28D) in the presence (dark bars) and absence (light bars) of RimJ overexpression. Data are presented as normalized values and are an average of at least three independent experiments. Units of normalized β-galactosidase activity for the ΔlacZ S5(G28D) strain with various pSG constructs (Kirthi et al., 2006) and p-rimJ are indicated as ratios to those observed in the isogenic MC252 strain.

RimJ associates with assembling 30S subunits in vivo. A. Analysis of ribosomal profile of S5(G28D) grown at the non-permissive temperature with induction of p-rimJ. Fractions from across this gradient were analysed by (I) slot-blot for RimJ or (II) gel electrophoresis for 23S rRNA and 16S rRNA to determine location of ribosomal particles. B. Primer extension analysis of 16S rRNAs extracted from immunoprecipitated RNPs with pBAD (Lanes 1, 4), non-induced p-rimJ (Lanes 2, 5) and induced p-rimJ (Lanes 3, 6) in S5(G28D) cells. Ni-NTA beads were washed with B-150 buffer (Lanes 1–3) or imidazole (Lanes 4–6). C. Western analysis of RNPs immunoprecipitated with pBAD (Lane 1), non-induced p-rimJ (Lane 2) and induced p-rimJ (Lane 3) in S5(G28D) cells for S4 and RimJ-V5 (indicated by arrows). D. Modified in vitro assembly map for 30S subunits (Grondek and Culver, 2004). Arrows indicate dependency between components for association with 16S rRNA. Positions of S4, S5 and S3 are indicated by circles.