Mutational Analysis of S12 Protein and Implications for the Accuracy of Decoding by the Ribosome

Divya Sharma, Anthony R. Cukras, Elizabeth J. Rogers, Daniel R. Southworth, Rachel Green

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)1065-1076
Number of pages12
JournalJournal of Molecular Biology
Volume374
Issue number4
DOIs
StatePublished - Dec 7 2007

Fingerprint

Paromomycin
Transfer RNA
Ribosomes
16S Ribosomal RNA
Proteins
Small Ribosome Subunits
Aminoglycosides
Streptomycin
Nucleotides
Binding Sites
Phenotype
Mutation

Keywords

  • decoding
  • domain closure
  • miscoding
  • paromomycin
  • S12

ASJC Scopus subject areas

  • Virology

Cite this

Mutational Analysis of S12 Protein and Implications for the Accuracy of Decoding by the Ribosome. / Sharma, Divya; Cukras, Anthony R.; Rogers, Elizabeth J.; Southworth, Daniel R.; Green, Rachel.

In: Journal of Molecular Biology, Vol. 374, No. 4, 07.12.2007, p. 1065-1076.

Research output: Contribution to journalArticle

Sharma, Divya ; Cukras, Anthony R. ; Rogers, Elizabeth J. ; Southworth, Daniel R. ; Green, Rachel. / Mutational Analysis of S12 Protein and Implications for the Accuracy of Decoding by the Ribosome. In: Journal of Molecular Biology. 2007 ; Vol. 374, No. 4. pp. 1065-1076.
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