Ribosomal protein L15 as a probe of 50 S ribosomal subunit structure.
L15, a 15 kDa protein of the large ribosomal subunit, interacts with over ten other proteins during 50 S assembly in vitro. We have probed the interaction L15 with 23 S rRNA in 50 S ribosomal subunits by chemical footprinting, and have used localized hydroxyl radical probing, generated from Fe(II) tethered to unique sites of L15, to characterize the three-dimensional 23 S rRNA environment of L15. Footprinting of L15 was done by reconstituting purified, recombinant L15 with core particles derived from Escherichia coli 50 S subunits by treatment with 2 M LiCl. The cores migrate as compact 50 S-like particles in sucrose gradients, contain 23 S and 5 S rRNA, and lack a subset of the 50 S proteins, including L15. Using both Fe(II).EDTA and dimethyl sulfate, we have identified a strong footprint for L15 in the region spanning nucleotides 572-654 in domain II of 23 S rRNA. This footprint cannot be detected when L15 is incubated with "naked" 23 S rRNA, indicating that formation of the L15 binding site requires a partially assembled particle.Protein-tethered hydroxyl radical probing was done using mutants of L15 containing single cysteine residues at amino acid positions 68, 71 and 115. The mutant proteins were derivatized with 1-[p-(bromo-acetamido)benzyl]-EDTA. Fe(II), bound to core particles, and hydroxyl radical cleavage was initiated. Distinct but overlapping sets of cleavages were obtained in the footprinted region of domain II, and in specific regions of domains I, IV and V of 23 S rRNA. These data locate L15 in proximity to several 23 S rRNA elements that are dispersed in the secondary structure, consistent with its central role in the latter stages of 50 S subunit assembly. Furthermore, these results indicate the proximity of these rRNA regions to one another, providing constraints on the tertiary folding of 23 S rRNA.
Lieberman
K R
KR
Center for the Molecular Biology of RNA, University of California, Santa Cruz, CA, 95064, USA.
Noller
H F
HF
D004492:Edetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D017665:Hydroxyl Radical / Q000737:chemistry; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007502:Iron Chelating Agents / Q000737:chemistry; D018021:Lithium Chloride / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009942:Organometallic Compounds / Q000737:chemistry; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism*
D007502:Iron Chelating Agents; D009942:Organometallic Compounds; D012338:RNA, Ribosomal, 23S; D012269:Ribosomal Proteins; C043649:ribosomal protein L15; D017665:Hydroxyl Radical; C042007:1-(4-bromoacetamidobenzyl)EDTA; D004492:Edetic Acid; D007501:Iron; D018021:Lithium Chloride