Modulation of decoding fidelity by ribosomal proteins S4 and S5

Abstract

Ribosomal proteins S4 and S5 participate in the decoding and assembly processes on the ribosome and the interaction with specific antibiotic inhibitors of translation. Many of the characterized mutations affecting these proteins decrease the accuracy of translation, leading to a ribosomal-ambiguity phenotype. Structural analyses of ribosomal complexes indicate that the tRNA selection pathway involves a transition between the closed and open conformations of the 30S ribosomal subunit and requires disruption of the interface between the S4 and S5 proteins. In agreement with this observation, several of the mutations that promote miscoding alter residues located at the S4-S5 interface. Here, the Escherichia coli rpsD and rpsE genes encoding the S4 and S5 proteins were targeted for mutagenesis and screened for accuracy-altering mutations. While a majority of the 38 mutant proteins recovered decrease the accuracy of translation, error-restrictive mutations were also recovered; only a minority of the mutant proteins affected rRNA processing, ribosome assembly, or interactions with antibiotics. Several of the mutations affect residues at the S4-S5 interface. These include five nonsense mutations that generate C-terminal truncations of S4. These truncations are predicted to destabilize the S4-S5 interface and, consistent with the domain closure model, all have ribosomal-ambiguity phenotypes. A substantial number of the mutations alter distant locations and conceivably affect tRNA selection through indirect effects on the S4-S5 interface or by altering interactions with adjacent ribosomal proteins and 16S rRNA.

FIG 1

Effects of selected mutations in ribosomal proteins S5 and S4 on growth on solid medium at 20°C, 37°C, and 42°C. Three-microliter aliquots of serially diluted (10⁻² to 10⁻⁶) overnight cultures of the mutants indicated were spotted onto antibiotic-free LB plates and incubated for 24 to 48 h. WT, wild type.

FIG 2

Effects of altered ribosomal proteins S4 and S5 on missense decoding (top) and UGA readthrough (bottom). Missense decoding values were obtained from pEK7-transformed cells and reflect near-cognate decoding of AAU by the AAA/G-decoding tRNA^Lys. Misreading levels are expressed in RLU as described in the text. Readthrough levels were obtained from strains transformed with lacZ plasmid pSG3/4 UGA, carrying a UGA mutation in the 5′ end of the β-galactosidase coding region. Bars represent (Miller) units of β-galactosidase activity (43). +Tyr-Val indicates a C-terminal extension of S4. WT, wild type.

FIG 3

Ribosome profiles of a wild-type strain and selected S4 and S5 mutants that displayed altered profiles. The positions of 30S and 50S subunits and 70S ribosomes are indicated on the profile of the wild-type strain, and the direction of sedimentation is from left to right.

FIG 4

rRNA processing in wild-type (WT) and selected S4 and S5 mutant strains. Total RNAs from logarithmically growing wild-type and mutant strains were extracted, electrophoresed on agarose-synergel gels, and stained with ethidium bromide. The second lane from the left contains RNAs extracted from wild-type cells treated with chloramphenicol for 30 min, leading to the accumulation of unprocessed 17S rRNA precursors (20, 21).

FIG 5

Prediction of structural effects of amino acid substitutions and termination mutants on the basis of the wild-type E. coli 70S ribosome crystal structure (; PDB entries 2AVY and 2AW4). In all of the panels, S4 is blue, S5 is red, other ribosomal proteins are dark gray, and the 16S rRNA is light gray. (A) Surface representation of the solvent side of the 30S subunit showing the relative locations of ribosomal proteins S4 (pale green) and S5 (light blue). For orientation, the 50S subunit is shown as a transparent surface behind the 30S subunit. (B) Sites of amino acid substitutions in S4 and S5 identified in this study. The α carbons of residues mutated in restrictive and ram mutants are shown as yellow and red spheres, respectively. (C) Sites of substitutions potentially affecting the S4-S5 interface either directly by changing residues involved at the interface or indirectly by affecting the local S4 or S5 conformation. Positions in S4 from E165 to the C terminus are dark green, including the C-terminal α-helix at the S4-S5 interface. Mutated residues are shown as sticks. The 16S rRNA is white. (D) Proximity of 16S rRNA residues to the S4-S5 interface. (E) Sites of mutations potentially affecting S4-16S rRNA interactions distant from the S4-S5 interface. (F) Sites of mutations potentially affecting S5-16S rRNA interactions distant from the S4-S5 interface. The N134Y substitution affects the processing of the 16S rRNA.