Protein-RNA Dynamics in the Central Junction Control 30S Ribosome Assembly

J Mol Biol. 2016 Sep 11;428(18):3615-31. doi: 10.1016/j.jmb.2016.05.010. Epub 2016 May 15.


Interactions between ribosomal proteins (rproteins) and ribosomal RNA (rRNA) facilitate the formation of functional ribosomes. S15 is a central domain primary binding protein that has been shown to trigger a cascade of conformational changes in 16S rRNA, forming the functional structure of the central domain. Previous biochemical and structural studies in vitro have revealed that S15 binds a three-way junction of helices 20, 21, and 22, including nucleotides 652-654 and 752-754. All junction nucleotides except 653 are highly conserved among the Bacteria. To identify functionally important motifs within the junction, we subjected nucleotides 652-654 and 752-754 to saturation mutagenesis and selected and analyzed functional mutants. Only 64 mutants with greater than 10% ribosome function in vivo were isolated. S15 overexpression complemented mutations in the junction loop in each of the partially active mutants, although mutations that produced inactive ribosomes were not complemented by overexpression of S15. Single-molecule Förster or fluorescence resonance energy transfer (smFRET) was used to study the Mg(2+)- and S15-induced conformational dynamics of selected junction mutants. Comparison of the structural dynamics of these mutants with the wild type in the presence and absence of S15 revealed specific sequence and structural motifs in the central junction that are important in ribosome function.

Keywords: 16S rRNA; RNA–protein interactions; ribosome protein S15; saturation mutagenesis; smFRET.

MeSH terms

  • DNA Mutational Analysis
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Fluorescence Resonance Energy Transfer
  • Genetic Complementation Test
  • Macromolecular Substances / metabolism*
  • Magnesium / metabolism
  • Models, Biological
  • Models, Molecular
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Maps
  • RNA, Ribosomal, 16S / chemistry
  • RNA, Ribosomal, 16S / genetics
  • RNA, Ribosomal, 16S / metabolism*
  • Ribosomal Proteins / metabolism*
  • Ribosome Subunits, Small, Bacterial / metabolism*


  • Macromolecular Substances
  • RNA, Ribosomal, 16S
  • Ribosomal Proteins
  • ribosomal protein S15
  • Magnesium