The C-terminus of ribosomal protein uS4 contributes to small ribosomal subunit biogenesis and the fidelity of translation

Abstract

Ribosomal protein uS4 is an essential ribosomal component involved in multiple functions, including mRNA decoding. Structural analyses indicate that during decoding, the interface between the C-terminus of uS4 and protein uS5 is disrupted and in agreement with this, C-terminal uS4 truncation mutants are readily isolated on the basis of their increased miscoding phenotypes. The same mutants can also display defects in small subunit assembly and 16S rRNA processing and some are temperature sensitive for growth. Starting with one such temperature sensitive Escherichia coli uS4 mutant, we have isolated temperature insensitive derivatives carrying additional, intragenic mutations that restore the C-terminus and ameliorate the ribosomal defects. At least one of these suppressors has no detectable ribosome biogenesis phenotype, yet still miscodes, suggesting that the C-terminal requirements for ribosome assembly are less rigid than for mRNA decoding. In contrast to the uS4 C-terminal mutants that increase miscoding, two Salmonella enterica uS4 mutants with altered C-termini have been reported as being error-restrictive. Here, reconstruction experiments demonstrate that contrary to the previous reports, these mutants have a distinct error-prone, increased misreading phenotype, consistent with the behavior of the equivalent E. coli mutants and their likely structural effects on uS4-uS5 interactions.

Keywords: Intragenic suppressors; Ribosomal protein uS4; Ribosome; Ribosome assembly; Translational accuracy; rRNA processing.

Figure 1

Location of ribosomal proteins uS4 and uS5 on the 70S ribosome and positions of mutations affecting uS4. The left panel depicts a surface rendering of the E. coli 70S ribosome based on the 3.5 Å crystal structure (pdb entry 2AVY; [31]). The 30S subunit is shown as light grey (16S rRNA) and dark grey (ribosomal proteins). Ribosomal proteins uS5 and uS4 are colored skyblue and palegreen, respectively. (Right panel) Cartoon rendering of ribosomal protein uS4 (palegreen) and its contact with ribosomal protein uS5 (skyblue). The region of uS4 affected by mutations (residues 166:205) is colored red and the α-carbons of every tenth residue from 160 to 200 are shown as spheres and numbered.

Figure 2

Growth of uS4 mutants on solid medium. Overnight cultures of the indicated strains were serially-diluted and 2.5 μl aliquots were spotted onto LB plates that were incubated at 20 °C (left panel), 37 °C (middle panel), or 42 °C (right panel) for 24–72 hrs. WT = wild type. A dagger (†) denotes the presence of the E62K rpsQ mutation affecting ribosomal protein S17 in the indicated strains.

Figure 3

Effects of alterations in ribosomal protein uS4 on UGA (right panel) and UAG (left panel) readthrough. Wild type and mutant cells were transformed with ampicillin resistant, lacZ reporter plasmids carrying either in-frame UGA (p34-11) or UAG (p12-6) stop codons and assayed for β-galactosidase activity. Cells were grown at either 37 °C (solid black bars), or 42 °C (stippled grey bars) and β-galactosidase activities are expressed in Miller units. Each bar represents the average activity of at least 3 independent samples (± SE).

Figure 4

Ribosome profiles of wild type and uS4 mutant strains grown at 42°C. Elution of free subunits, 70S ribosomes and polysomes was detected by monitoring the A₂₆₀ (y-axis) with an ISCO UV6 detector. The positions of 30S and 50S subunits and 70S ribosomes are indicated on selected gradients.

Figure 5

Processing of rRNA in wild type and uS4 mutant cells grown at 42°C. Total RNAs extracted from logarithmically growing cells were electrophoresed on agarose-Synergel gels and stained with ethidium bromide. The second lane from the left contains RNAs from wild type cells treated with chloramphenicol, which inhibits 16S rRNA processing.

Figure 6

Stop codon readthrough in wild type E. coli and S. enterica strains expressing wild type or mutant uS4 proteins. Bars represent the β-galactosidase activities (expressed in Miller units) of E. coli (EC) or S. enterica (Sal) strains expressing the indicated uS4 proteins and transformed with the UAG lacZ reporter plasmid p12-6 (left panel) or the UGA reporter, p34-11 (right panel). Each bar reflects the average activity of at least 3 independent samples (± SE).