The 23 S rRNA environment of ribosomal protein L9 in the 50 S ribosomal subunit.
Ribosomal protein L9 consists of two globular alpha/beta domains separated by a nine-turn alpha-helix. We examined the rRNA environment of L9 by chemical footprinting and directed hydroxyl radical probing. We reconstituted L9, or individual domains of L9, with L9-deficient 50 S subunits, or with deproteinized 23 S rRNA. A footprint was identified in domain V of 23 S rRNA that was mainly attributable to N-domain binding. Fe(II) was tethered to L9 via cysteine residues introduced at positions along the alpha-helix and in the C-domain, and derivatized proteins were reconstituted with L9-deficient subunits. Directed hydroxyl radical probing targeted regions of domains I, III, IV, and V of 23 S rRNA, reinforcing the view that 50 S subunit architecture is typified by interwoven rRNA domains. There was a striking correlation between the cleavage patterns from the Fe(II) probes attached to the alpha-helix and their predicted orientations, constraining both the position and orientation of L9, as well as the arrangement of specific elements of 23 S rRNA, in the 50 S subunit.
Lieberman
K R
KR
Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California, Santa Cruz, CA, 95064, USA.
Firpo
M A
MA
Herr
A J
AJ
Nguyenle
T
T
Atkins
J F
JF
Gesteland
R F
RF
Noller
H F
HF
D001665:Binding Sites; D004492:Edetic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005296:Ferrous Compounds / Q000378:metabolism; D005818:Genetic Engineering; D017665:Hydroxyl Radical / Q000378:metabolism; D008958:Models, Molecular; D015335:Molecular Probes / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D017433:Protein Structure, Secondary; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013463:Sulfuric Acid Esters / Q000378:metabolism
D005296:Ferrous Compounds; D015335:Molecular Probes; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; D013463:Sulfuric Acid Esters; C049650:ribosomal protein L9; C055526:Fe(II)-EDTA; D017665:Hydroxyl Radical; D004492:Edetic Acid; C007482:dimethyl sulfate