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
.