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. 1998 Jan;4(1):112–123.

In vitro selection and characterization of streptomycin-binding RNAs: recognition discrimination between antibiotics.

S T Wallace 1, R Schroeder 1
PMCID: PMC1369601  PMID: 9436913

Abstract

As pathogens continue to evade therapeutical drugs, a better understanding of the mode of action of antibiotics continues to have high importance. A growing body of evidence points to RNA as a crucial target for antibacterial and antiviral drugs. For example, the aminocyclitol antibiotic streptomycin interacts with the 16S ribosomal RNA and, in addition, inhibits group I intron splicing. To understand the mode of binding of streptomycin to RNA, we isolated small, streptomycin-binding RNA aptamers via in vitro selection. In addition, bluensomycin, a streptomycin analogue that does not inhibit splicing, was used in a counter-selection to obtain RNAs that bind streptomycin with high affinity and specificity. Although an RNA from the normal selection (motif 2) bound both antibiotics, an RNA from the counter-selection (motif 1) discriminated between streptomycin and bluensomycin by four orders of magnitude. The binding site of streptomycin on the RNAs was determined via chemical probing with dimethylsulfate and kethoxal. The minimal size required for drug binding was a 46- and a 41-mer RNA for motifs 1 and 2, respectively. Using Pb2+ cleavage in the presence and absence of streptomycin, a conformational change spanning the entire mapped sequence length of motif 1 was observed only when both streptomycin and Mg2+ were present. Both RNAs require Mg2+ for binding streptomycin.

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Selected References

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  1. Bass B. L., Cech T. R. Ribozyme inhibitors: deoxyguanosine and dideoxyguanosine are competitive inhibitors of self-splicing of the Tetrahymena ribosomal ribonucleic acid precursor. Biochemistry. 1986 Aug 12;25(16):4473–4477. doi: 10.1021/bi00364a001. [DOI] [PubMed] [Google Scholar]
  2. Bass B. L., Cech T. R. Specific interaction between the self-splicing RNA of Tetrahymena and its guanosine substrate: implications for biological catalysis by RNA. 1984 Apr 26-May 2Nature. 308(5962):820–826. doi: 10.1038/308820a0. [DOI] [PubMed] [Google Scholar]
  3. Cech T. R., Szewczak A. A. Selecting apt RNAs for NMR. RNA. 1996 Jul;2(7):625–627. [PMC free article] [PubMed] [Google Scholar]
  4. DAVIES J., GILBERT W., GORINI L. STREPTOMYCIN, SUPPRESSION, AND THE CODE. Proc Natl Acad Sci U S A. 1964 May;51:883–890. doi: 10.1073/pnas.51.5.883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davies J., Gorini L., Davis B. D. Misreading of RNA codewords induced by aminoglycoside antibiotics. Mol Pharmacol. 1965 Jul;1(1):93–106. [PubMed] [Google Scholar]
  6. Davies J. Structure-activity relationships among the aminoglycoside antibiotics. Antimicrob Agents Chemother (Bethesda) 1967;7:297–303. [PubMed] [Google Scholar]
  7. Eaton B. E., Gold L., Zichi D. A. Let's get specific: the relationship between specificity and affinity. Chem Biol. 1995 Oct;2(10):633–638. doi: 10.1016/1074-5521(95)90023-3. [DOI] [PubMed] [Google Scholar]
  8. Famulok M., Hüttenhofer A. In vitro selection analysis of neomycin binding RNAs with a mutagenized pool of variants of the 16S rRNA decoding region. Biochemistry. 1996 Apr 9;35(14):4265–4270. doi: 10.1021/bi952479r. [DOI] [PubMed] [Google Scholar]
  9. Fourmy D., Recht M. I., Blanchard S. C., Puglisi J. D. Structure of the A site of Escherichia coli 16S ribosomal RNA complexed with an aminoglycoside antibiotic. Science. 1996 Nov 22;274(5291):1367–1371. doi: 10.1126/science.274.5291.1367. [DOI] [PubMed] [Google Scholar]
  10. Gornicki P., Baudin F., Romby P., Wiewiorowski M., Kryzosiak W., Ebel J. P., Ehresmann C., Ehresmann B. Use of lead(II) to probe the structure of large RNA's. Conformation of the 3' terminal domain of E. coli 16S rRNA and its involvement in building the tRNA binding sites. J Biomol Struct Dyn. 1989 Apr;6(5):971–984. doi: 10.1080/07391102.1989.10506525. [DOI] [PubMed] [Google Scholar]
  11. Hendrix M., Priestley E. S., Joyce G. F., Wong C. H. Direct observation of aminoglycoside-RNA interactions by surface plasmon resonance. J Am Chem Soc. 1997 Apr 23;119(16):3641–3648. doi: 10.1021/ja964290o. [DOI] [PubMed] [Google Scholar]
  12. Jenison R. D., Gill S. C., Pardi A., Polisky B. High-resolution molecular discrimination by RNA. Science. 1994 Mar 11;263(5152):1425–1429. doi: 10.1126/science.7510417. [DOI] [PubMed] [Google Scholar]
  13. Jiang L., Suri A. K., Fiala R., Patel D. J. Saccharide-RNA recognition in an aminoglycoside antibiotic-RNA aptamer complex. Chem Biol. 1997 Jan;4(1):35–50. doi: 10.1016/s1074-5521(97)90235-0. [DOI] [PubMed] [Google Scholar]
  14. Lato S. M., Boles A. R., Ellington A. D. In vitro selection of RNA lectins: using combinatorial chemistry to interpret ribozyme evolution. Chem Biol. 1995 May;2(5):291–303. doi: 10.1016/1074-5521(95)90048-9. [DOI] [PubMed] [Google Scholar]
  15. Melançon P., Lemieux C., Brakier-Gingras L. A mutation in the 530 loop of Escherichia coli 16S ribosomal RNA causes resistance to streptomycin. Nucleic Acids Res. 1988 Oct 25;16(20):9631–9639. doi: 10.1093/nar/16.20.9631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Michel F., Hanna M., Green R., Bartel D. P., Szostak J. W. The guanosine binding site of the Tetrahymena ribozyme. Nature. 1989 Nov 23;342(6248):391–395. doi: 10.1038/342391a0. [DOI] [PubMed] [Google Scholar]
  17. Moazed D., Noller H. F. Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature. 1987 Jun 4;327(6121):389–394. doi: 10.1038/327389a0. [DOI] [PubMed] [Google Scholar]
  18. Montandon P. E., Wagner R., Stutz E. E. coli ribosomes with a C912 to U base change in the 16S rRNA are streptomycin resistant. EMBO J. 1986 Dec 20;5(13):3705–3708. doi: 10.1002/j.1460-2075.1986.tb04703.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. PESTKA S., MARSHALL R., NIRENBERG M. RNA CODEWORDS AND PROTEIN SYNTHESIS. V. EFFECT OF STREPTOMYCIN ON THE FORMATION OF RIBOSOME-SRNA COMPLEXES. Proc Natl Acad Sci U S A. 1965 Mar;53:639–646. doi: 10.1073/pnas.53.3.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Purohit P., Stern S. Interactions of a small RNA with antibiotic and RNA ligands of the 30S subunit. Nature. 1994 Aug 25;370(6491):659–662. doi: 10.1038/370659a0. [DOI] [PubMed] [Google Scholar]
  21. Schroeder R., von Ahsen U., Belfort M. Effects of mutations of the bulged nucleotide in the conserved P7 pairing element of the phage T4 td intron on ribozyme function. Biochemistry. 1991 Apr 2;30(13):3295–3303. doi: 10.1021/bi00227a018. [DOI] [PubMed] [Google Scholar]
  22. Sjögren A. S., Pettersson E., Sjöberg B. M., Strömberg R. Metal ion interaction with cosubstrate in self-splicing of group I introns. Nucleic Acids Res. 1997 Feb 1;25(3):648–653. doi: 10.1093/nar/25.3.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stern S., Moazed D., Noller H. F. Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. Methods Enzymol. 1988;164:481–489. doi: 10.1016/s0076-6879(88)64064-x. [DOI] [PubMed] [Google Scholar]
  24. Thierfelder W. E., van Deursen J. M., Yamamoto K., Tripp R. A., Sarawar S. R., Carson R. T., Sangster M. Y., Vignali D. A., Doherty P. C., Grosveld G. C. Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells. Nature. 1996 Jul 11;382(6587):171–174. doi: 10.1038/382171a0. [DOI] [PubMed] [Google Scholar]
  25. Wallis M. G., Streicher B., Wank H., von Ahsen U., Clodi E., Wallace S. T., Famulok M., Schroeder R. In vitro selection of a viomycin-binding RNA pseudoknot. Chem Biol. 1997 May;4(5):357–366. doi: 10.1016/s1074-5521(97)90126-5. [DOI] [PubMed] [Google Scholar]
  26. Wallis M. G., von Ahsen U., Schroeder R., Famulok M. A novel RNA motif for neomycin recognition. Chem Biol. 1995 Aug;2(8):543–552. doi: 10.1016/1074-5521(95)90188-4. [DOI] [PubMed] [Google Scholar]
  27. Wang J. F., Cech T. R. Tertiary structure around the guanosine-binding site of the Tetrahymena ribozyme. Science. 1992 Apr 24;256(5056):526–529. doi: 10.1126/science.1315076. [DOI] [PubMed] [Google Scholar]
  28. Wang Y., Rando R. R. Specific binding of aminoglycoside antibiotics to RNA. Chem Biol. 1995 May;2(5):281–290. doi: 10.1016/1074-5521(95)90047-0. [DOI] [PubMed] [Google Scholar]
  29. Wank H., Rogers J., Davies J., Schroeder R. Peptide antibiotics of the tuberactinomycin family as inhibitors of group I intron RNA splicing. J Mol Biol. 1994 Mar 4;236(4):1001–1010. doi: 10.1016/0022-2836(94)90007-8. [DOI] [PubMed] [Google Scholar]
  30. Yarus M. A specific amino acid binding site composed of RNA. Science. 1988 Jun 24;240(4860):1751–1758. doi: 10.1126/science.3381099. [DOI] [PubMed] [Google Scholar]
  31. Zimmermann G. R., Jenison R. D., Wick C. L., Simorre J. P., Pardi A. Interlocking structural motifs mediate molecular discrimination by a theophylline-binding RNA. Nat Struct Biol. 1997 Aug;4(8):644–649. doi: 10.1038/nsb0897-644. [DOI] [PubMed] [Google Scholar]
  32. Zuker M. On finding all suboptimal foldings of an RNA molecule. Science. 1989 Apr 7;244(4900):48–52. doi: 10.1126/science.2468181. [DOI] [PubMed] [Google Scholar]
  33. von Ahsen U., Noller H. F. Footprinting the sites of interaction of antibiotics with catalytic group I intron RNA. Science. 1993 Jun 4;260(5113):1500–1503. doi: 10.1126/science.8502993. [DOI] [PubMed] [Google Scholar]
  34. von Ahsen U., Schroeder R. Streptomycin inhibits splicing of group I introns by competition with the guanosine substrate. Nucleic Acids Res. 1991 May 11;19(9):2261–2265. doi: 10.1093/nar/19.9.2261. [DOI] [PMC free article] [PubMed] [Google Scholar]

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