Following the dynamics of changes in solvent accessibility of 16 S and 23 S rRNA during ribosomal subunit association using synchrotron-generated hydroxyl radicals.
We have probed the structure and dynamics of ribosomal RNA in the Escherichia coli ribosome using equilibrium and time-resolved hydroxyl radical (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 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 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 OH footprinting studies, the reactivity profiles obtained using Fe(II)EDTA and X-ray generated OH were comprehensively compared. The reactivity patterns are similar except for a small number of nucleotides that have decreased reactivity to 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 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.
Nguyenle
Thuylinh
T
Center for Molecular Biology of RNA and Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, 95064, USA.
Laurberg
Martin
M
Brenowitz
Michael
M
Noller
Harry F
HF
D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D017665:Hydroxyl Radical / Q000737:chemistry*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000737:chemistry*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry*; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry*; D013499:Surface Properties; D017356:Synchrotrons; D016963:Thermus thermophilus; D013997:Time Factors; D017186:Titrimetry
D009711:Nucleotides; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D012997:Solvents; D017665:Hydroxyl Radical