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. 1984 Apr 11;12(7):3405–3423. doi: 10.1093/nar/12.7.3405

Mapping of psoralen cross-linked nucleotides in RNA.

E Garrett-Wheeler, R E Lockard, A Kumar
PMCID: PMC318754  PMID: 6425802

Abstract

A method is described for using the cross-linking reagent 4'-(hydroxy-methyl)-4,5',8-trimethylpsoralen (HMT) to map base paired regions and higher-order structure within RNA molecules. Applying this method to yeast tRNAPhe, we have specifically identified cross-links within the acceptor stem between U6 X U68, in the D-stem between C11 X C25, and in the T psi-stem between U50 X C63 and U52 X C63. We have also identified a unique cross-link between U8 X C48 which are trans pyrimidines in the core region due to tertiary interactions between U8:A14 and C48:G15. The precise point of cross-linking was deduced in every case by using purine-specific U2 ribonuclease along with cytidine-specific CL3 ribonuclease which will anomalously cleave after photoreversed pyrimidines. The ability to map the precise point of cross-linking should prove invaluable in identifying nucleotides in close proximity within the tertiary structure of other RNA molecules.

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Selected References

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

  1. Bachellerie J. P., Hearst J. E. Specificity of the photoreaction of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen with ribonucleic acid. Identification of reactive sites in Escherichia coli phenylalanine-accepting transfer ribonucleic acid. Biochemistry. 1982 Mar 16;21(6):1357–1363. doi: 10.1021/bi00535a039. [DOI] [PubMed] [Google Scholar]
  2. Boguski M. S., Hieter P. A., Levy C. C. Identification of a cytidine-specific ribonuclease from chicken liver. J Biol Chem. 1980 Mar 10;255(5):2160–2163. [PubMed] [Google Scholar]
  3. Chatterjee P. K., Cantor C. R. Photochemical production of psoralen - DNA monoadducts capable of subsequent photocrosslinking. Nucleic Acids Res. 1978 Oct;5(10):3619–3633. doi: 10.1093/nar/5.10.3619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Favorova O. O., Fasiolo F., Keith G., Vassilenko S. K., Ebel J. P. Partial digestion of tRNA--aminoacyl-tRNA synthetase complexes with cobra venom ribonuclease. Biochemistry. 1981 Feb 17;20(4):1006–1011. doi: 10.1021/bi00507a055. [DOI] [PubMed] [Google Scholar]
  5. Hassur S. M., Whitlock H. W., Jr UV shadowing--a new and convenient method for the location of ultraviolet-absorbing species in polyacrylamide gels. Anal Biochem. 1974 May;59(1):162–164. doi: 10.1016/0003-2697(74)90020-7. [DOI] [PubMed] [Google Scholar]
  6. Johnson R. A., Walseth T. F. The enzymatic preparation of [alpha-32P]ATP, [alpha-32P]GTP, [32P]cAMP, and [32P]cGMP, and their use in the assay of adenylate and guanylate cyclases and cyclic nucleotide phosphodiesterases. Adv Cyclic Nucleotide Res. 1979;10:135–167. [PubMed] [Google Scholar]
  7. Lockard R. E., Alzner-Deweerd B., Heckman J. E., MacGee J., Tabor M. W., RajBhandary U. L. Sequence analysis of 5'[32P] labeled mRNA and tRNA using polyacrylamide gel electrophoresis. Nucleic Acids Res. 1978 Jan;5(1):37–56. doi: 10.1093/nar/5.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lockard R. E., Connaughton J. F., Kumar A. Nucleotide sequence of the 5'- and 3'- domains for rabbit 18S ribosomal RNA. Nucleic Acids Res. 1982 Jun 11;10(11):3445–3457. doi: 10.1093/nar/10.11.3445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lockard R. E., Kumar A. Mapping tRNA structure in solution using double-strand-specific ribonuclease V1 from cobra venom. Nucleic Acids Res. 1981 Oct 10;9(19):5125–5140. doi: 10.1093/nar/9.19.5125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Peattie D. A., Gilbert W. Chemical probes for higher-order structure in RNA. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4679–4682. doi: 10.1073/pnas.77.8.4679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Quigley G. J., Rich A. Structural domains of transfer RNA molecules. Science. 1976 Nov 19;194(4267):796–806. doi: 10.1126/science.790568. [DOI] [PubMed] [Google Scholar]
  12. Rabin D., Crothers D. M. Analysis of RNA secondary structure by photochemical reversal of psoralen crosslinks. Nucleic Acids Res. 1979 Oct 10;7(3):689–703. doi: 10.1093/nar/7.3.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Shapiro D. J. Quantitative ethanol precipitation of nanogram quantities of DNA and RNA. Anal Biochem. 1981 Jan 1;110(1):229–231. doi: 10.1016/0003-2697(81)90139-1. [DOI] [PubMed] [Google Scholar]
  14. Thompson J. F., Bachellerie J. P., Hall K., Hearst J. E. Dependence of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen photoaddition on the conformation of ribonucleic acid. Biochemistry. 1982 Mar 16;21(6):1363–1368. doi: 10.1021/bi00535a040. [DOI] [PubMed] [Google Scholar]
  15. Thompson J. F., Hearst J. E. Structure of E. coli 16S RNA elucidated by psoralen crosslinking. Cell. 1983 Apr;32(4):1355–1365. doi: 10.1016/0092-8674(83)90316-1. [DOI] [PubMed] [Google Scholar]
  16. Thompson J. F., Wegnez M. R., Hearst J. E. Determination of the secondary structure of Drosophila melanogaster 5 S RNA by hydroxymethyltrimethylpsoralen crosslinking. J Mol Biol. 1981 Apr 15;147(3):417–436. doi: 10.1016/0022-2836(81)90493-9. [DOI] [PubMed] [Google Scholar]
  17. Turner S., Noller H. F. Identification of sites of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen cross-linking in Escherichia coli 23S ribosomal ribonucleic acid. Biochemistry. 1983 Aug 16;22(17):4159–4164. doi: 10.1021/bi00286a026. [DOI] [PubMed] [Google Scholar]
  18. Turner S., Thompson J. F., Hearst J. E., Noller H. F. Identification of a site of psoralen crosslinking in E. coli 16S ribosomal RNA. Nucleic Acids Res. 1982 May 11;10(9):2839–2849. doi: 10.1093/nar/10.9.2839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wollenzien P. L., Cantor C. R. Gel electrophoretic technique for separating crosslinked RNAs. Application to improved electron microscopic analysis of psoralen crosslinked 16 S ribosomal RNA. J Mol Biol. 1982 Jul 25;159(1):151–166. doi: 10.1016/0022-2836(82)90036-5. [DOI] [PubMed] [Google Scholar]
  20. Wrede P., Wurst R., Vournakis J., Rich A. Conformational changes of yeast tRNAPhe and E. coli tRNA2Glu as indicated by different nuclease digestion patterns. J Biol Chem. 1979 Oct 10;254(19):9608–9616. [PubMed] [Google Scholar]
  21. Wurst R. M., Vournakis J. N., Maxam A. M. Structure mapping of 5'-32P-labeled RNA with S1 nuclease. Biochemistry. 1978 Oct 17;17(21):4493–4499. doi: 10.1021/bi00614a021. [DOI] [PubMed] [Google Scholar]

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