title: Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome.
abstract: The fidelity of aminoacyl-tRNA selection by the ribosome depends on a conformational switch in the decoding center of the small ribosomal subunit induced by cognate but not by near-cognate aminoacyl-tRNA. The aminoglycosides paromomycin and streptomycin bind to the decoding center and induce related structural rearrangements that explain their observed effects on miscoding. Structural and biochemical studies have identified ribosomal protein S12 (as well as specific nucleotides in 16S ribosomal RNA) as a critical molecular contributor in distinguishing between cognate and near-cognate tRNA species as well as in promoting more global rearrangements in the small subunit, referred to as "closure." Here we use a mutational approach to define contributions made by two highly conserved loops in S12 to the process of tRNA selection. Most S12 variant ribosomes tested display increased levels of fidelity (a "restrictive" phenotype). Interestingly, several variants, K42A and R53A, were substantially resistant to the miscoding effects of paromomycin. Further characterization of the compromised paromomycin response identified a probable second, fidelity-modulating binding site for paromomycin in the 16S ribosomal RNA that facilitates closure of the small subunit and compensates for defects associated with the S12 mutations.
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Divya
authors.initials: D
authors.lastname: Sharma
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Cukras
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Rogers
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Southworth
authors.forename: Rachel
authors.initials: R
authors.lastname: Green
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010303:Paromomycin / Q000494:pharmacology; D014176:Protein Biosynthesis; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000502:physiology*; D016415:Sequence Alignment; D013307:Streptomycin / Q000494:pharmacology
chemical_list: D029968:Escherichia coli Proteins; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins; C506766:RpsL protein, E coli; D010303:Paromomycin; D013307:Streptomycin