Suppression of a cold‐sensitive mutation in ribosomal protein S5 reveals a role for RimJ in ribosome biogenesis

Abstract

SummaryA specific mutation of Escherichia coli ribosomal protein S5, in which glycine is changed to aspartate at position 28 [S5(G28D)], results in cold sensitivity and defects in ribosome biogenesis and translational fidelity. In an attempt to understand the roles of S5 in these essential cellular functions, we selected extragenic suppressors and identified rimJ as a high-copy suppressor of the cold-sensitive phenotype associated with the S5(G28D) mutation. Our studies indicate that RimJ overexpression suppresses the growth defects, anomalous ribosome profiles and mRNA misreading exhibited by the S5(G28D) mutant strain. Although previously characterized as the N-acetyltransferase of S5, our data indicate that RimJ, when devoid of acetyltransferase activity, can suppress S5(G28D) defects thus indicating that the suppression activity of RimJ is not dependent on its acetyltransferase activity. Additionally, RimJ appears to associate with pre-30S subunits indicating that it acts on the ribonucleoprotein particle. These findings suggest that RimJ has evolved dual functionality; it functions in r-protein acetylation and as a ribosome assembly factor in E. coli.

Appropriate maturation and folding of 16S rRNA during 30S subunit biogenesis are critical for translational fidelity

“…As stated above, in earlier work we demonstrated that RimJ, when overexpressed in the S5(G28D) background, mitigated the fidelity errors evident in the S5(G28D) strain (11). Therefore, a role of RimJ in rescuing 16S rRNA processing defects of the S5 (G28D) strain was assessed.…”

“…1C). An increase in mature 16S rRNA in 70S ribosomes correlates with increased translational fidelity in the S5(G28D) background, yet RimJ does not associate with translating ribosomes (11).…”

“…Cells were grown at 37°C (permissive temperature) to ∼0.7 OD or grown at 37°C to ∼0.2 OD prior to being shifted to 20°C, which is the nonpermissive temperature for S5 (G28D) cells or room temperature for ΔrpsO cells and incubated for another 4 h. RimJ expression in S5(G28D) strain was done as described earlier (11). Ribosome profiles were essentially performed as described (37), with sedimentation adjusted to allow separation of SSUs and pre-SSUs at the expense of observing polysomes.…”

“…Interestingly, a single extragenic high copy suppressor of the coldsensitive phenotype associated with S5(G28D) was isolated and was identified to be rimJ (11), the gene that encodes S5 N-terminal acetyltransferase (12). However, acetylation of S5 is not a factor in the observed suppression as an acetyltransferasedeficient form of RimJ is sufficient to suppress the phenotypic changes associated with S5(G28D) (11). Although it might be expected that this suppressor would allow the decoupling of the biogenesis and fidelity effects associated with S5(G28D), overexpression of RimJ rescued not only biogenesis associated defects observed in this S5 mutant strain, but also rescued the miscoding phenotype near to WT levels (11).…”

“…However, acetylation of S5 is not a factor in the observed suppression as an acetyltransferasedeficient form of RimJ is sufficient to suppress the phenotypic changes associated with S5(G28D) (11). Although it might be expected that this suppressor would allow the decoupling of the biogenesis and fidelity effects associated with S5(G28D), overexpression of RimJ rescued not only biogenesis associated defects observed in this S5 mutant strain, but also rescued the miscoding phenotype near to WT levels (11). Hence, we hypothesized that there is a previously unexplored link between SSU biogenesis and translational fidelity and RimJ rescues the fidelity defects of S5(G28D) by rescuing the SSU biogenesis defects of the S5 mutant strain.…”