In vivo selection of spectinomycin-binding RNAs.

The folding of even short RNA molecules in a random library can produce a huge number of possible macromolecular structures. Using this principle, we have designed selections to seek non-coding RNA transcripts capable of interfering with specific macromolecules such as transcription factors in living bacterial cells. Here we show that such selections can uncover an unexpected class of RNAs. In the present case, we report short RNA transcripts whose expression confers bacterial resistance to the antibiotic spectinomycin. We provide evidence that such RNAs cause drug resistance by direct antibiotic binding, demonstrating a class of spectinomycin-specific functional molecular decoys built from RNA.

[1]  Poul Nissen,et al.  Placement of protein and RNA structures into a 5 Å-resolution map of the 50S ribosomal subunit , 1999, Nature.

[2]  F. Whipple.  Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli. , 1998, Nucleic acids research.

[3]  L. J. Maher,et al.  Combinatorial selection of a small RNA that induces amplification of IncFII plasmids in Escherichia coli. , 1998, Journal of molecular biology.

[4]  G. Soukup,et al.  Selection and characterization of RNAs that relieve transcriptional interference in Escherichia coli. , 1998, Nucleic acids research.

[5]  C. Prescott,et al.  The selection in vivo and characterization of an RNA recognition motif for spectinomycin. , 1997, Bioorganic & medicinal chemistry.

[6]  Harry F. Noller,et al.  Interaction of antibiotics with functional sites in 16S ribosomal RNA , 1987, Nature.

[7]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[8]  A. Tocilj,et al.  The small ribosomal subunit from Thermus thermophilus at 4.5 A resolution: pattern fittings and the identification of a functional site. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[9]  T. Earnest,et al.  X-ray crystal structures of 70S ribosome functional complexes. , 1999, Science.

[10]  R. W. Davis,et al.  Genetic selection for genes encoding sequence-specific DNA-binding proteins. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. Ehrenberg,et al.  Ribosomal RNA and protein mutants resistant to spectinomycin. , 1990, The EMBO journal.

[12]  L. J. Maher,et al.  Selection and Characterization of an RNA Decoy for Transcription Factor NF-κB† , 1999 .

[13]  L. J. Maher,et al.  Recognition of duplex DNA by RNA polynucleotides. , 1995, Nucleic acids research.

[14]  H. Noller,et al.  Independent in vitro assembly of a ribonucleoprotein particle containing the 3' domain of 16S rRNA. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  V. Ramakrishnan,et al.  Structure of a bacterial 30S ribosomal subunit at 5.5 Å resolution , 1999, Nature.

[16]  V. Ramakrishnan,et al.  Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics , 2000, Nature.

[17]  J. Sabina,et al.  Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. , 1999, Journal of molecular biology.

[18]  V. Ramakrishnan,et al.  The Structural Basis for the Action of the Antibiotics Tetracycline, Pactamycin, and Hygromycin B on the 30S Ribosomal Subunit , 2000, Cell.

[19]  L. Cassiday,et al.  In vivo recognition of an RNA aptamer by its transcription factor target. , 2001, Biochemistry.

[20]  P. Moore.  The ribosome at atomic resolution. , 2001, Biochemistry.