Ribosomal protein L15 as a probe of 50 S ribosomal subunit structure

Citation
Kr. Lieberman et Hf. Noller, Ribosomal protein L15 as a probe of 50 S ribosomal subunit structure, J MOL BIOL, 284(5), 1998, pp. 1367-1378
Citations number
46
Categorie Soggetti
Molecular Biology & Genetics
Journal title
JOURNAL OF MOLECULAR BIOLOGY
ISSN journal
00222836 → ACNP
Volume
284
Issue
5
Year of publication
1998
Pages
1367 - 1378
Database
ISI
SICI code
0022-2836(199812)284:5<1367:RPLAAP>2.0.ZU;2-G
Abstract
L15, a 15 kDa protein of the large ribosomal subunit, interacts with over t en other proteins during 50 S assembly in vitro. We have probed the interac tion L15 with 23 S rRNA in 50 S ribosomal subunits by chemical footprinting , and have used localized hydroxyl radical probing, generated from Fe(II) t ethered 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 wi th core particles derived from Escherichia coil 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 protein s, including L15. Using both Fe(II) EDTA and dimethyl sulfate, we have iden tified a strong footprint for L15 in the region spanning nucleotides 572-65 4 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 bind ing site requires a partially assembled particle. Protein-tethered hydroxyl radical probing was done using mutants of L15 con taining single cysteine residues at amino acid positions 68, 71 and 115. Th e mutant proteins were derivatized with 1-[p-(bromo-acetamido)benzyl]-EDTA Fe(II), bound to core particles, and hydroxyl radical cleavage was initiate d. Distinct but overlapping sets of cleavages were obtained in the foot-pri nted 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 element s that are dispersed in the secondary structure, consistent with its centra l role in the latter stages of 50 S subunit assembly. Furthermore, these re sults indicate the proximity of these rRNA regions to one another, providin g constraints on the tertiary folding of 23 S rRNA. (C) 1998 Academic Press .