Directed hydroxyl radical probing of the rRNA neighborhood of ribosomal protein S13 using tethered Fe(II).
Directed hydroxyl radical probing was used to probe the rRNA neighborhood around protein S13 in the 30S ribosomal subunit. The unique cysteine at position 84 of S13 served as a tethering site for attachment of Fe(II)-1-(p-bromoacetamidobenzyl)-EDTA. Derivatized S13 (Fe-C84-S13) was then assembled into 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins. Hydroxyl radicals generated from the tethered Fe(II) resulted in cleavage of the RNA backbone in two localized regions of the 3' major domain of 16S rRNA. One region spans nt 1308-1333 and is close to a site previously crosslinked to S13. A second set of cleavages is found in the 950/1230 helix. Both regions have been implicated in binding of S13 by previous chemical footprinting studies using base-specific chemical probes and solution-based hydroxyl radical probing. These results place both regions of 16S rRNA in proximity to position C84 of S13 in the three-dimensional structure of the 30S ribosomal subunit.
Heilek
G M
GM
Center for the Molecular Biology of RNA, Sinsheimer Laboratories, University of California, Santa Cruz 95064, USA.
Noller
H F
HF
D001426:Bacterial Proteins / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004492:Edetic Acid / Q000031:analogs & derivatives; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D006861:Hydrogen Peroxide; D017665:Hydroxyl Radical; D007501:Iron; D008958:Models, Molecular; D015335:Molecular Probes; D009690:Nucleic Acid Conformation; D009942:Organometallic Compounds; D012329:RNA, Bacterial / Q000737:chemistry*; D012335:RNA, Ribosomal / Q000737:chemistry*; D012269:Ribosomal Proteins / Q000737:chemistry*; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D015335:Molecular Probes; D009942:Organometallic Compounds; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C040469:ribosomal protein S30; C512838:rpsM protein, E coli; D017665:Hydroxyl Radical; C042007:1-(4-bromoacetamidobenzyl)EDTA; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D007501:Iron; D003545:Cysteine