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

Ribosomal protein S5 is critical for small ribosomal subunit (SSU) assembly and is indispensable for SSU function. Previously, we identified a point mutation in S5, (G28D) that alters both SSU formation and translational fidelity in vivo, which is unprecedented for other characterized S5 mutations. Surprisingly, additional copies of an extraribosomal assembly factor, RimJ, rescued all the phenotypes associated with S5(G28D), including fidelity defects, suggesting that the effect of RimJ on rescuing the miscoding of S5(G28D) is indirect. To understand the underlying mechanism, we focused on the biogenesis cascade and observed defects in processing of precursor 16S (p16S) rRNA in the S5(G28D) strain, which were rescued by RimJ. Analyses of p16S rRNA-containing ribosomes from other strains further supported a correspondence between the extent of 5(') end maturation of 16S rRNA and translational miscoding. Chemical probing of mutant ribosomes with additional leader sequences at the 5(') end of 16S rRNA compared to WT ribosomes revealed structural differences in the region of helix 1. Thus, the presence of additional nucleotides at the 5(') end of 16S rRNA could alter fidelity by changing the architecture of 16S rRNA in translating ribosomes and suggests that fidelity is governed by accuracy and completeness of the SSU biogenesis cascade.

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

A 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.

Assembly of the 30S Ribosomal Subunit.

Protein synthesis involves nearly a third of the total molecules in a typical bacterial cell. Within the cell, protein synthesis is performed by the ribosomes, and research over several decades has investigated ribosomal formation, structure, and function. This review provides an overview of the current understanding of the assembly of the Escherichia coli 30S ribosomal subunit. The E. coli 30S subunit contains one rRNA molecule (16S) and 21 ribosomal proteins (r-proteins; S1 to S21). The formation of functional subunits can occur as a self-assembly process in vitro; i.e., all the information required for the formation of active ribosomes resides in the primary sequences of the r-proteins and rRNAs. In vitro reconstitution of functional 30S subunits is carried out by using a mixture of TP30, individually purified natural or recombinant r-proteins, and natural 16S rRNA. Chemical probing and primer extension analysis have been used extensively to monitor changes in the reactivities of nucleotides in 16S rRNA during the in vitro reconstitution of 30S subunits. The potential roles for r-proteins in 30S subunit assembly were determined by omitting single proteins in reconstitution experiments. The RNPs resulting from single protein omissions were examined in terms of their composition and function to determine the roles of the absent proteins. Recent developments in understanding the structure of the 30S subunit have led to speculation about roles for some of the r-proteins in assembly. The crystal structures of the 30S subunit (1, 2) and the 70S ribosome (3) reveal details of the r-protein and rRNA interactions.

A novel single amino acid change in small subunit ribosomal protein S5 has profound effects on translational fidelity.

S5 is a small subunit ribosomal protein (r-protein) linked to the functional center of the 30S ribosomal subunit. In this study we have identified a unique amino acid mutation in Escherichia coli S5 that produces spectinomycin-resistance and cold sensitivity. This mutation significantly alters cell growth, folding of 16S ribosomal RNA, and translational fidelity. While translation initiation is not affected, both +1 and -1 frameshifting and nonsense suppression are greatly enhanced in the mutant strain. Interestingly, this S5 ribosome ambiguity-like mutation is spatially remote from previously identified S5 ribosome ambiguity (ram) mutations. This suggests that the mechanism responsible for ram phenotypes in the novel mutant strain is possibly distinct from those proposed for other known S5 (and S4) ram mutants. This study highlights the importance of S5 in ribosome function and cell physiology, and suggests that translational fidelity can be regulated in multiple ways.