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.

[1]  Ronald R. Breaker,et al.  Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP , 1999, Nature Structural Biology.

[2]  R. Rando,et al.  Specificity in the binding of aminoglycosides to HIV-RRE RNA. , 1999, Biochemistry.

[3]  R. Schroeder,et al.  Modulation of RNA function by aminoglycoside antibiotics , 2000, The EMBO journal.

[4]  Michael R. Green,et al.  Small molecules that selectively block RNA binding of HIV-1 rev protein inhibit rev function and viral production , 1993, Cell.

[5]  M. Nomura,et al.  Assembly Mapping of 30S Ribosomal Proteins from E. coli , 1970, Nature.

[6]  E. Westhof,et al.  Aminoglycoside-RNA interactions. , 1999, Current opinion in chemical biology.

[7]  M. Famulok,et al.  Oligonucleotide aptamers that recognize small molecules. , 1999, Current opinion in structural biology.

[8]  O. Uhlenbeck,et al.  Inhibition of the hammerhead ribozyme by neomycin. , 1969, RNA.

[9]  P. Hagerman,et al.  Protein and Mg(2+)-induced conformational changes in the S15 binding site of 16 S ribosomal RNA. , 1998, Journal of molecular biology.

[10]  Chi‐Huey Wong,et al.  Design of Small Molecules That Recognize RNA: Development of Aminoglycosides as Potential Antitumor Agents That Target Oncogenic RNA Sequences , 2000 .

[11]  J. Davies,et al.  Antibiotic inhibition of group I ribozyme function , 1991, Nature.

[12]  R R Breaker,et al.  Altering molecular recognition of RNA aptamers by allosteric selection. , 2000, Journal of molecular biology.

[13]  B. Ballou,et al.  Assembly mapping of 30 S ribosomal proteins from Escherichia coli. Further studies. , 1974, The Journal of biological chemistry.

[14]  Taekjip Ha,et al.  Mg2+-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[15]  J. Killian,et al.  Minimal RNA constructs that specifically bind aminoglycoside antibiotics with high affinities. , 1998, Biochemistry.

[16]  A D Ellington,et al.  Design and optimization of effector-activated ribozyme ligases. , 2000, Nucleic acids research.

[17]  J W Szostak,et al.  Isolation and characterization of fluorophore-binding RNA aptamers. , 1998, Folding & design.

[18]  X. Zhuang,et al.  Ligand-induced conformational changes observed in single RNA molecules. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[19]  A. Pyle,et al.  Site-specific labeling of RNA with fluorophores and other structural probes. , 1999, Methods.

[20]  J. Williamson,et al.  Structure of the S15,S6,S18-rRNA complex: assembly of the 30S ribosome central domain. , 2000, Science.

[21]  Chi-Huey Wong,et al.  Design of Bifunctional Antibiotics that Target Bacterial rRNA and Inhibit Resistance-Causing Enzymes , 2000 .

[22]  R. Rando,et al.  Specific binding of aminoglycoside antibiotics to RNA. , 1995, Chemistry & biology.

[23]  R. Rando,et al.  The binding site of a specific aminoglycoside binding RNA molecule. , 1998, Biochemistry.

[24]  G. Varani,et al.  Recent solution structures of RNA and its complexes with drugs, peptides and proteins. , 1997, Current opinion in structural biology.

[25]  R. Breaker,et al.  Engineering precision RNA molecular switches. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[26]  R R Breaker,et al.  Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes. , 2001, RNA.

[27]  J. Puglisi,et al.  Structure of the A Site of Escherichia coli 16S Ribosomal RNA Complexed with an Aminoglycoside Antibiotic , 1996, Science.