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. 1985 Mar;82(5):1381–1385. doi: 10.1073/pnas.82.5.1381

Mapping the location of psoralen crosslinks on RNA by mung bean nuclease sensitivity of RNA.DNA hybrids.

C F Hui, C R Cantor
PMCID: PMC397265  PMID: 2983344

Abstract

An indirect high resolution method has been developed for finding the location of intrastrand crosslinks in RNA. An end-labeled DNA strand that overlaps the approximate crosslink position is hybridized to the RNA and then treated with mung bean nuclease. The resulting digest is analyzed on a sequencing-type gel. The method was tested with the major psoralen crosslink seen in the 16S rRNA of inactivated Escherichia coli 30S ribosomal subunits. This crosslink was previously mapped between residues 930 +/- 25 and a region close to the 3' end by electron microscopy. The new indirect method reveals that the crosslink occurs between residues 919 and 923 and residues 1530 and 1534. When these results are examined in the light of existing consensus secondary structure models for the 16S rRNA, it appears that the Shine-Dalgarno sequence is located close to the peptidyl tRNA binding site.

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Selected References

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

  1. Amils R., Matthews E. A., Cantor C. R. Reconstitution of 50 S ribosomal subunits from Escherichia coli. Methods Enzymol. 1979;59:449–461. doi: 10.1016/0076-6879(79)59107-1. [DOI] [PubMed] [Google Scholar]
  2. Barnes W. M. DNA sequencing by partial ribosubstitution. J Mol Biol. 1978 Feb 15;119(1):83–99. doi: 10.1016/0022-2836(78)90271-1. [DOI] [PubMed] [Google Scholar]
  3. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  4. Berk A. J., Sharp P. A. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. doi: 10.1073/pnas.75.3.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brosius J., Palmer M. L., Kennedy P. J., Noller H. F. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4801–4805. doi: 10.1073/pnas.75.10.4801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brosius J., Ullrich A., Raker M. A., Gray A., Dull T. J., Gutell R. R., Noller H. F. Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli. Plasmid. 1981 Jul;6(1):112–118. doi: 10.1016/0147-619x(81)90058-5. [DOI] [PubMed] [Google Scholar]
  7. Carbon P., Ebel J. P., Ehresmann C. The sequence of the ribosomal 16S RNA from Proteus vulgaris. Sequence comparison with E. coli 16S RNA and its use in secondary model building. Nucleic Acids Res. 1981 May 25;9(10):2325–2333. doi: 10.1093/nar/9.10.2325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Casey J., Davidson N. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 1977;4(5):1539–1552. doi: 10.1093/nar/4.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garrett-Wheeler E., Lockard R. E., Kumar A. Mapping of psoralen cross-linked nucleotides in RNA. Nucleic Acids Res. 1984 Apr 11;12(7):3405–3423. doi: 10.1093/nar/12.7.3405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gupta R., Lanter J. M., Woese C. R. Sequence of the 16S Ribosomal RNA from Halobacterium volcanii, an Archaebacterium. Science. 1983 Aug 12;221(4611):656–659. doi: 10.1126/science.221.4611.656. [DOI] [PubMed] [Google Scholar]
  11. Kroeker W. D., Kowalski D. Gene-sized pieces produced by digestion of linear duplex DNA with mung bean nuclease. Biochemistry. 1978 Aug 8;17(16):3236–3243. doi: 10.1021/bi00609a010. [DOI] [PubMed] [Google Scholar]
  12. Kroeker W. D., Kowalski D., Laskowski M., Sr Mung bean nuclease I. Terminally directed hydrolysis of native DNA. Biochemistry. 1976 Oct 5;15(20):4463–4467. doi: 10.1021/bi00665a020. [DOI] [PubMed] [Google Scholar]
  13. Marko M. A., Chipperfield R., Birnboim H. C. A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal Biochem. 1982 Apr;121(2):382–387. doi: 10.1016/0003-2697(82)90497-3. [DOI] [PubMed] [Google Scholar]
  14. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  15. Noller H. F., Woese C. R. Secondary structure of 16S ribosomal RNA. Science. 1981 Apr 24;212(4493):403–411. doi: 10.1126/science.6163215. [DOI] [PubMed] [Google Scholar]
  16. Prince J. B., Taylor B. H., Thurlow D. L., Ofengand J., Zimmermann R. A. Covalent crosslinking of tRNA1Val to 16S RNA at the ribosomal P site: identification of crosslinked residues. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5450–5454. doi: 10.1073/pnas.79.18.5450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rubtsov P. M., Musakhanov M. M., Zakharyev V. M., Krayev A. S., Skryabin K. G., Bayev A. A. The structure of the yeast ribosomal RNA genes. I. The complete nucleotide sequence of the 18S ribosomal RNA gene from Saccharomyces cerevisiae. Nucleic Acids Res. 1980 Dec 11;8(23):5779–5794. doi: 10.1093/nar/8.23.5779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Salim M., Maden B. E. Nucleotide sequence of Xenopus laevis 18S ribosomal RNA inferred from gene sequence. Nature. 1981 May 21;291(5812):205–208. doi: 10.1038/291205a0. [DOI] [PubMed] [Google Scholar]
  19. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Steitz J. A., Jakes K. How ribosomes select initiator regions in mRNA: base pair formation between the 3' terminus of 16S rRNA and the mRNA during initiation of protein synthesis in Escherichia coli. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4734–4738. doi: 10.1073/pnas.72.12.4734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Thammana P., Cantor C. R., Wollenzien P. L., Hearst J. E. Crosslinking studies on the organization of the 16 S ribosomal RNA within the 30 S Escherichia coli ribosomal subunit. J Mol Biol. 1979 Nov 25;135(1):271–283. doi: 10.1016/0022-2836(79)90352-8. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Woese C. R., Gutell R., Gupta R., Noller H. F. Detailed analysis of the higher-order structure of 16S-like ribosomal ribonucleic acids. Microbiol Rev. 1983 Dec;47(4):621–669. doi: 10.1128/mr.47.4.621-669.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Wollenzien P., Hearst J. E., Thammana P., Cantor C. R. Base-pairing between distant regions of the Escherichia coli 16 S ribosomal RNA in solution. J Mol Biol. 1979 Nov 25;135(1):255–269. doi: 10.1016/0022-2836(79)90351-6. [DOI] [PubMed] [Google Scholar]

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