Following the Dynamics of Changes in Solvent Accessibility of 16 S and 23 S rRNA During Ribosomal Subunit Association Using Synchrotron-generated Hydroxyl Radicals

Thuylinh Nguyenle, Martin Laurberg, Michael Brenowitz, Harry F. Noller

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

We have probed the structure and dynamics of ribosomal RNA in the Escherichia coli ribosome using equilibrium and time-resolved hydroxyl radical ({radical dot}OH) RNA footprinting to explore changes in the solvent-accessible surface of the rRNA with single-nucleotide resolution. The goal of these studies is to better understand the structural transitions that accompany association of the 30 S and 50 S subunits and to build a foundation for the quantitative analysis of ribosome structural dynamics during translation. Clear portraits of the subunit interface surfaces for 16 S and 23 S rRNA were obtained by constructing difference maps between the {radical dot}OH protection maps of the free subunits and that of the associated ribosome. In addition to inter-subunit contacts consistent with the crystal structure, additional {radical dot}OH protections are evident in regions at or near the subunit interface that reflect association-induced conformational changes. Comparison of these data with the comparable difference maps of the solvent-accessible surface of the rRNA calculated for the Thermus thermophilus X-ray crystal structures shows extensive agreement but also distinct differences. As a prelude to time-resolved {radical dot}OH footprinting studies, the reactivity profiles obtained using Fe(II)EDTA and X-ray generated {radical dot}OH were comprehensively compared. The reactivity patterns are similar except for a small number of nucleotides that have decreased reactivity to {radical dot}OH generated from Fe(II)EDTA compared to X-rays. These nucleotides are generally close to ribosomal proteins, which can quench diffusing radicals by virtue of side-chain oxidation. Synchrotron X-ray {radical dot}OH footprinting was used to monitor the kinetics of association of the 30 S and 50 S subunits. The rates individually measured for the inter-subunit contacts are comparable within experimental error. The application of this approach to the study of ribosome dynamics during the translation cycle is discussed.

Original languageEnglish (US)
Pages (from-to)1235-1248
Number of pages14
JournalJournal of Molecular Biology
Volume359
Issue number5
DOIs
StatePublished - Jun 23 2006

Keywords

  • association kinetics
  • chemical probing
  • intersubunit bridges
  • ribosomes
  • subunit interface

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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