A three-fluorophore FRET assay for high-throughput screening of small-molecule inhibitors of ribosome assembly.
In one of the first steps of prokaryotic ribosome assembly, the ribosomal protein S15 binds to a three-way junction in the central domain of the 16S rRNA. Binding causes a conformational change that is required for subsequent binding events. Using a novel fluorescence resonance energy transfer assay with three fluorophores, two on the RNA and one on the S15 protein, small-molecule libraries can be screened for potential inhibitors of this initial step in ribosome assembly. The employment of three fluorophores allows both the conformational change of the RNA and the binding of S15 to be monitored in a single assay.
Klostermeier
Dagmar
D
The Scripps Research Institute, Department of Molecular Biology, La Jolla, CA 92037, USA.
Sears
Pamela
P
Wong
Chi-Huey
CH
Millar
David P
DP
Williamson
James R
JR
D000617:Aminoglycosides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D008274:Magnesium / Q000378:metabolism; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000617:Aminoglycosides; D005456:Fluorescent Dyes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins; C036004:ribosomal protein S15; D008274:Magnesium