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. 1991 Jul 11;19(13):3653–3660. doi: 10.1093/nar/19.13.3653

Conformation and structural fluctuations of a 218 nucleotides long rRNA fragment: 4-thiouridine as an intrinsic photolabelling probe.

Y L Dubreuil 1, A Expert-Bezançon 1, A Favre 1
PMCID: PMC328394  PMID: 1712940

Abstract

The structure in solution of an RNA fragment (218 nucleotides long) containing part of E. coli 16S rRNA domain 2 has been studied using the intrinsic photoaffinity probe 4-thiouridine (s4U). In vitro transcription with T7 polymerase, in the presence of s4U triphosphate yielded complete RNA molecules. An affinity electrophoresis system based on Phenylmercuric substituted polyacrylamide (APM) gels allows separation of the RNA chains as a function of their s4U content. Distribution of s4U within chains follows a binomial law indicating that (i) substitution is close to random, (ii) efficiency of s4U incorporation is 0.22 times that of U. The monothiolated RNA fraction isolated from APM gel was irradiated at 366 nm under native conditions and the intramolecularly crosslinked molecules, (34%), were separated on denaturing polyacrylamide gel according to loop size. The positions of the two partners of bridges were identified by mean of reverse transcription and RNA sequencing. 17 of the 41 possible s4U positions lead to detectable bridges. These crosslinks formed efficiently at the border of bihelical regions or when structural mobility is allowed. The pattern of crosslinks is in agreement with the previously proposed secondary structure but indicates that it is much more flexible than expected.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brimacombe R., Atmadja J., Stiege W., Schüler D. A detailed model of the three-dimensional structure of Escherichia coli 16 S ribosomal RNA in situ in the 30 S subunit. J Mol Biol. 1988 Jan 5;199(1):115–136. doi: 10.1016/0022-2836(88)90383-x. [DOI] [PubMed] [Google Scholar]
  2. Cramer F., Gottschalk E. M., Matzura H., Scheit K. H., Sternbach H. The synthesis of the alternating copolymer poly(r(A-s4U)) by RNA polymerase of Escherichia coli. Eur J Biochem. 1971 Apr;19(3):379–385. doi: 10.1111/j.1432-1033.1971.tb01327.x. [DOI] [PubMed] [Google Scholar]
  3. Ericson G., Wollenzien P. Use of reverse transcription to determine the exact locations of psoralen photochemical crosslinks in RNA. Anal Biochem. 1988 Oct;174(1):215–223. doi: 10.1016/0003-2697(88)90538-6. [DOI] [PubMed] [Google Scholar]
  4. Favre A., Bezerra R., Hajnsdorf E., Lemaigre Dubreuil Y., Expert-Bezançon A. Substitution of uridine in vivo by the intrinsic photoactivable probe 4-thiouridine in Escherichia coli RNA. Its use for E. coli ribosome structural analysis. Eur J Biochem. 1986 Nov 3;160(3):441–449. doi: 10.1111/j.1432-1033.1986.tb10060.x. [DOI] [PubMed] [Google Scholar]
  5. Gregory R. J., Cahill P. B., Thurlow D. L., Zimmermann R. A. Interaction of Escherichia coli ribosomal protein S8 with its binding sites in ribosomal RNA and messenger RNA. J Mol Biol. 1988 Nov 20;204(2):295–307. doi: 10.1016/0022-2836(88)90577-3. [DOI] [PubMed] [Google Scholar]
  6. Igloi G. L. Interaction of tRNAs and of phosphorothioate-substituted nucleic acids with an organomercurial. Probing the chemical environment of thiolated residues by affinity electrophoresis. Biochemistry. 1988 May 17;27(10):3842–3849. doi: 10.1021/bi00410a048. [DOI] [PubMed] [Google Scholar]
  7. Kean J. M., Draper D. E. Secondary structure of a 345-base RNA fragment covering the S8/S15 protein binding domain of Escherichia coli 16S ribosomal RNA. Biochemistry. 1985 Sep 10;24(19):5052–5061. doi: 10.1021/bi00340a015. [DOI] [PubMed] [Google Scholar]
  8. Moazed D., Noller H. F. Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes. Cell. 1986 Dec 26;47(6):985–994. doi: 10.1016/0092-8674(86)90813-5. [DOI] [PubMed] [Google Scholar]
  9. Mougel M., Ehresmann B., Ehresmann C. Binding of Escherichia coli ribosomal protein S8 to 16S rRNA: kinetic and thermodynamic characterization. Biochemistry. 1986 May 20;25(10):2756–2765. doi: 10.1021/bi00358a003. [DOI] [PubMed] [Google Scholar]
  10. Mougel M., Eyermann F., Westhof E., Romby P., Expert-Bezançon A., Ebel J. P., Ehresmann B., Ehresmann C. Binding of Escherichia coli ribosomal protein S8 to 16 S rRNA. A model for the interaction and the tertiary structure of the RNA binding site. J Mol Biol. 1987 Nov 5;198(1):91–107. doi: 10.1016/0022-2836(87)90460-8. [DOI] [PubMed] [Google Scholar]
  11. Mougel M., Philippe C., Ebel J. P., Ehresmann B., Ehresmann C. The E. coli 16S rRNA binding site of ribosomal protein S15: higher-order structure in the absence and in the presence of the protein. Nucleic Acids Res. 1988 Apr 11;16(7):2825–2839. doi: 10.1093/nar/16.7.2825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Prescott C. D., Göringer H. U. A single mutation in 16S rRNA that affects mRNA binding and translation-termination. Nucleic Acids Res. 1990 Sep 25;18(18):5381–5386. doi: 10.1093/nar/18.18.5381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Scheit K. G. Die Synthese von 4-Thiouridin-5'-diphosphat, 4-Thiouridin-5'-triphosphat und Desoxy-4-thiothymidin-5'-triphosphat. Chem Ber. 1968;101(4):1141–1147. doi: 10.1002/cber.19681010402. [DOI] [PubMed] [Google Scholar]
  14. Silberklang M., Gillum A. M., RajBhandary U. L. Use of in vitro 32P labeling in the sequence analysis of nonradioactive tRNAs. Methods Enzymol. 1979;59:58–109. doi: 10.1016/0076-6879(79)59072-7. [DOI] [PubMed] [Google Scholar]
  15. Tate W., Greuer B., Brimacombe R. Codon recognition in polypeptide chain termination: site directed crosslinking of termination codon to Escherichia coli release factor 2. Nucleic Acids Res. 1990 Nov 25;18(22):6537–6544. doi: 10.1093/nar/18.22.6537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Teare J., Wollenzien P. The structure of a pre-mRNA molecule in solution determined with a site directed cross-linking reagent. Nucleic Acids Res. 1990 Feb 25;18(4):855–864. doi: 10.1093/nar/18.4.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wollenzien P. L., Murphy R. F., Cantor C. R., Expert-Bezançon A., Hayes D. H. Structure of the Escherichia coli 16 S ribosomal RNA. Psoralen crosslinks and N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine crosslinks detected by electron microscopy. J Mol Biol. 1985 Jul 5;184(1):67–80. doi: 10.1016/0022-2836(85)90044-0. [DOI] [PubMed] [Google Scholar]
  18. Wollenzien P., Expert-Bezançon A., Favre A. Sites of contact of mRNA with 16S rRNA and 23S rRNA in the Escherichia coli ribosome. Biochemistry. 1991 Feb 19;30(7):1788–1795. doi: 10.1021/bi00221a009. [DOI] [PubMed] [Google Scholar]

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