Modulation of Decoding Fidelity by Ribosomal Proteins S4 and S5

Abstract

bRibosomal 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. C ellular protein synthesis systems translate mRNAs quickly and with high accuracy. The accuracy of the decoding process can, however, be altered by agents such as the antibiotic streptomycin that promote miscoding and by mutations in rRNA, ribosomal proteins, or translation factors (1). Among the first such accuracy mutants to be characterized were some of the streptomycin-resistant Escherichia coli strains carrying alterations in ribosomal protein S12 (2). Subsequently, E. coli mutants carrying altered ribosomal protein S4 or S5 were isolated that supported increased levels of miscoding (3-5). Since those early studies, many other mutants have been isolated that affect the accuracy of decoding and carry alterations in different components of the translation machinery (1, 4).The determination of high-resolution structures of ribosomes has offered structural interpretations of the effects of some accuracy-altering ribosomal mutations (6). X-ray crystallography of ribosomal complexes has shown that conserved RNA elements of the decoding center use a shape-sensing mechanism to monitor base pairing between the A site codon and the anticodon of incoming aminoacyl-tRNA. Successful interaction of a ternary complex of EF-Tu-GTP-aminoacyl-tRNA with mRNA in the decoding center triggers a series of conformational rearrangements of the head and shoulder domains of the 30S subunit, ultimately resulting in the formation of a ...

The Loop 2 Region of Ribosomal Protein uS5 Influences Spectinomycin Sensitivity, Translational Fidelity, and Ribosome Biogenesis

“…Mutagenesis of the rpsE gene was facilitated by the use of a strain carrying a kanamycin resistance cassette downstream of the rpsE coding region, as described previously (11). The GGT glycine codon corresponding to Gly27 in protein uS5 was targeted for mutagenesis in two separate experiments with two different oligonucleotides.…”

“…␤-Galactosidase assays were carried out on permeabilized, logarithmically growing cells, and activity was expressed in Miller units (11,45). Luciferase assays to test for missense errors were performed using the Dual-Luciferase Reporter assay system from Promega, and luminescence was measured with a GloMax 20/20 luminometer according to the manufacturers' instructions.…”

“…Amino acid substitutions potentially destabilizing the interface are predicted to alter tRNA selection pathways by favoring the transition to the closed conformation. In keeping with this interpretation, many of the classical, and some of the newly isolated, uS4 and uS5 miscoding mutants affect residues at the interface (11,12). However, the affected residues in some other uS4 and uS5 accuracy-altering mutants are distant from the interface, and it is unclear how they disrupt decoding.…”

Epistasis analysis of 16S rRNA ram mutations helps define the conformational dynamics of the ribosome that influence decoding

“…These include the h12/S4/S5 region, which lies on the solvent side of the subunit at an interface between the shoulder subdomain and the remainder of the subunit; h16, which represents the "top" of the shoulder; and h8/h14/L19; elements which contribute to intersubunit bridge B8 (Maisnier-Patin et al 2002McClory et al 2010). A recent reevaluation of S4/S5 mutations showed that all C-terminal truncations of S4 confer a ram phenotype (Agarwal et al 2015). These truncations undoubtedly destabilize the open conformation of the subunit, lending strong support to the idea that domain closure contributes to GTPase activation (Ogle and Ramakrishnan 2005;Voorhees et al 2010;Fredrick 2015).…”

Another Look at Mutations in Ribosomal Protein S4 Lends Strong Support to the Domain Closure Model

“…In this issue, Agarwal and coworkers (27) describe the results of a direct screen for S4-S5 mutations that alter translation fidelity in E. coli. Using a single-step recombineering approach, they mutagenized each gene specifically and screened for those mutations that increase or decrease miscoding.…”