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. 1979 Sep 25;7(2):465–480. doi: 10.1093/nar/7.2.465

Binding of magnesium ions and ethidium bromide: comparison of ribosomes and free ribosomal RNA.

D Elson, P Spitnik-Elson, S Avital, R Abramowitz
PMCID: PMC328029  PMID: 386285

Abstract

Comparative studies of free ribosomal RNA and ribosomes were made with two probes, Mg++ ions and ethidium bromide, which interact with RNA in different ways. Mg++. E. coli 16 S rRNA and 30 S ribosomes were equilibrated with four different buffers. Equilibration required several days at 4 degrees and several hours at 37 degrees. In all buffers ribosomes bound more Mg than free rRNA, the difference sometimes reaching 20--30%. Ribosomes were more resistant than free rRNA to heat denaturation and their denaturation was more highly cooperative. Ribosomes that bound more Mg++ had higher denaturation temperatures. Ethidium bromide. Fluorescence enhancement studies of ethidium intercalation showed the free 16 S rRNA to have 50--80 binding sites per molecule. A large fraction of these sites were present and accessible in the ribosome, but their ethidium-binding constants were reduced by an order of magnitude. In addition, free rRNA contained a small number of very strong binding sites that were virtually absent in the ribosomes.

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Selected References

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

  1. Aktipis S., Martz W. W. Circular dichroism and temperature--optical density studies on the conformation of polynucleotide--ethidium bromide complexes. Biochemistry. 1974 Jan 1;13(1):112–118. doi: 10.1021/bi00698a018. [DOI] [PubMed] [Google Scholar]
  2. Angerer L. M., Moudrianakis E. N. Interaction of ethidium bromide with whole and selectively deproteinized deoxynucleoproteins from calf thymus. J Mol Biol. 1972 Feb 14;63(3):505–521. doi: 10.1016/0022-2836(72)90444-5. [DOI] [PubMed] [Google Scholar]
  3. Araco A., Belli M., Giorgi C., Onori G. The secondary structure of E. coli ribosomes and ribosomal RNA's: a spectrophotometric approach. Nucleic Acids Res. 1975 Mar;2(3):373–381. doi: 10.1093/nar/2.3.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bittman R. Studies of the binding of ethidium bromide to transfer ribonucleic acid: absorption, fluorescence, ultracentrifugation and kinetic investigations. J Mol Biol. 1969 Dec 14;46(2):251–268. doi: 10.1016/0022-2836(69)90420-3. [DOI] [PubMed] [Google Scholar]
  5. Blake A., Peacocke A. R. The interaction of aminocridines with nucleic acids. Biopolymers. 1968;6(9):1225–1253. doi: 10.1002/bip.1968.360060902. [DOI] [PubMed] [Google Scholar]
  6. Bollen A., Herzog A., Favre A., Thibault J., Gros F. Fluorescence studies on the 30 S ribosome assembly process. FEBS Lett. 1970 Nov 9;11(1):49–54. doi: 10.1016/0014-5793(70)80489-6. [DOI] [PubMed] [Google Scholar]
  7. Borisova O. F., Molnar Ia, Samarina O. P. Iadernye ribonukleoproteidy, soderzhashchie informatsionnuiu RNK. 12. Izuchenie vtorichnoi struktury pro-mRNK v sostave iadernykh RNP-chastits fluorestsentnymi metodami. Mol Biol (Mosk) 1977 Mar-Apr;11(2):457–465. [PubMed] [Google Scholar]
  8. Brakier-Gingras L., Boileau G., Glorieux S., Brisson N. Streptomycin-induced conformational changes in the 70-S bacterial ribosome. Biochim Biophys Acta. 1978 Dec 21;521(2):413–425. doi: 10.1016/0005-2787(78)90283-6. [DOI] [PubMed] [Google Scholar]
  9. Choi Y. S., Carr C. W. Ion-binding studies of ribonucleic acid and Escherichia coli ribosomes. J Mol Biol. 1967 Apr 28;25(2):331–345. doi: 10.1016/0022-2836(67)90145-3. [DOI] [PubMed] [Google Scholar]
  10. Cox R. A., Pratt H., Huvos P., Higginson B., Hirst W. A study of the thermal stability of ribosomes and biologically active subribosomal particles. Biochem J. 1973 Jul;134(3):775–793. doi: 10.1042/bj1340775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ghysen A., Bollen A., Herzog A. Ionic effects on the ribosomal quaternary structure. Eur J Biochem. 1970 Mar 1;13(1):132–136. doi: 10.1111/j.1432-1033.1970.tb00908.x. [DOI] [PubMed] [Google Scholar]
  12. Goldberg A. Magnesium binding by Escherichia coli ribosomes. J Mol Biol. 1966 Feb;15(2):663–673. doi: 10.1016/s0022-2836(66)80134-1. [DOI] [PubMed] [Google Scholar]
  13. Hochkeppel H. K., Spicer E., Craven G. R. A method of preparing Escherichia coli 16 S RNA possessing previously unobserved 30 S ribosomal protein binding sites. J Mol Biol. 1976 Feb 25;101(2):155–170. doi: 10.1016/0022-2836(76)90369-7. [DOI] [PubMed] [Google Scholar]
  14. Kastrup R. V., Young M. A., Krugh T. R. Ethidium bromide complexes with self-complementary deoxytetranucleotides. Demonstration and discussion of sequence preferences in the intercalative binding of ethidium bromide. Biochemistry. 1978 Nov 14;17(23):4855–4865. doi: 10.1021/bi00616a002. [DOI] [PubMed] [Google Scholar]
  15. Kimes B. W., Morris D. R. Cations and ribosome structure. II. Effects on the 50S subunit of substituting polyamines for magnesium ion. Biochemistry. 1973 Jan 30;12(3):442–449. doi: 10.1021/bi00727a013. [DOI] [PubMed] [Google Scholar]
  16. Kreishman G. P., Chan S. I., Bauer W. Proton magnetic resonance study of the interaction of ethidium bromide with several uracil residues, uridylyl (3' leads to 5') uridine and polyuridylic acid. J Mol Biol. 1971 Oct 14;61(1):45–58. doi: 10.1016/0022-2836(71)90205-1. [DOI] [PubMed] [Google Scholar]
  17. LERMAN L. S. Structural considerations in the interaction of DNA and acridines. J Mol Biol. 1961 Feb;3:18–30. doi: 10.1016/s0022-2836(61)80004-1. [DOI] [PubMed] [Google Scholar]
  18. Le Pecq J. B. Use of ethidium bromide for separation and determination of nucleic acids of various conformational forms and measurement of their associated enzymes. Methods Biochem Anal. 1971;20:41–86. doi: 10.1002/9780470110393.ch2. [DOI] [PubMed] [Google Scholar]
  19. LePecq J. B., Paoletti C. A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterization. J Mol Biol. 1967 Jul 14;27(1):87–106. doi: 10.1016/0022-2836(67)90353-1. [DOI] [PubMed] [Google Scholar]
  20. Lurquin P. F., Seligy V. L. Binding of ethidium bromide to avian erythrocyte chromatin. Biochem Biophys Res Commun. 1972 Feb 16;46(3):1399–1404. doi: 10.1016/s0006-291x(72)80131-1. [DOI] [PubMed] [Google Scholar]
  21. Lurquin P., Buchet-Mahieu J. Biological activity of ethidium bromide - transfer RNA complexes. FEBS Lett. 1971 Jan 30;12(5):244–248. doi: 10.1016/0014-5793(71)80188-6. [DOI] [PubMed] [Google Scholar]
  22. Miall S. H., Walker I. O. Structural studies on ribosomes. II. Denaturation and sedimentation of ribosomal subunits unfolded in EDTA. Biochim Biophys Acta. 1969 Feb 18;174(2):551–560. doi: 10.1016/0005-2787(69)90284-6. [DOI] [PubMed] [Google Scholar]
  23. Miskin R., Zamir A., Elson D. Inactivation and reactivation of ribosomal subunits: the peptidyl transferase activity of the 50 s subunit of Escherihia coli. J Mol Biol. 1970 Dec 14;54(2):355–378. doi: 10.1016/0022-2836(70)90435-3. [DOI] [PubMed] [Google Scholar]
  24. Potapov A. P., Bogdanov A. A. Izuchenie kompaktnoi struktury maloi subchastitsy ribosom E. coli i ee RNK metodami fluorestsentnoi spektroskorii i skorostnoi sedimentatsii. Mol Biol (Mosk) 1977 May-Jun;11(3):545–554. [PubMed] [Google Scholar]
  25. SPIRIN A. S., KISELEV N. A., SHAKULOV R. S., BOGDANOV A. A. IZUCHENIE STRUKTURY RIBOSOM; OBRATIMOE RAZVORACHIVANIE RIBOSOMNYKH CHASTITS V RIBONUKLEOPROTEIDNYE TIAZHI I MODEL' UKLADKI. Biokhimiia. 1963 Sep-Oct;28:920–930. [PubMed] [Google Scholar]
  26. Sander C., Ts'o P. O. Interaction of nucleic acids. 8. Binding of magnesium ions by nucleic acids. J Mol Biol. 1971 Jan 14;55(1):1–21. doi: 10.1016/0022-2836(71)90276-2. [DOI] [PubMed] [Google Scholar]
  27. Sobell H. M., Tsai C. C., Jain S. C., Gilbert S. G. Visualization of drug-nucleic acid interactions at atomic resolution. III. Unifying structural concepts in understanding drug-DNA interactions and their broader implications in understanding protein-DNA interactions. J Mol Biol. 1977 Aug 15;114(3):333–365. doi: 10.1016/0022-2836(77)90254-6. [DOI] [PubMed] [Google Scholar]
  28. Spitnik-Elson P., Atsmon A. Detachment of ribosomal proteins by salt. I. Effect of conditions on the amount of protein detached. J Mol Biol. 1969 Oct 14;45(1):113–124. doi: 10.1016/0022-2836(69)90214-9. [DOI] [PubMed] [Google Scholar]
  29. Spitnik-Elson P., Elson D., Abramowitz R. A purified nucleoprotein fragment of the 30 S ribosomal subunit of Escherichia coli. Biochim Biophys Acta. 1979 Feb 27;561(2):435–444. doi: 10.1016/0005-2787(79)90151-5. [DOI] [PubMed] [Google Scholar]
  30. Spitnik-Elson P., Elson D., Abramowitz R., Avital S. A large nucleoprotein fragment of the 50-S ribosomal subunit of Escherichia coli. Biochim Biophys Acta. 1978 Nov 21;521(1):308–323. doi: 10.1016/0005-2787(78)90273-3. [DOI] [PubMed] [Google Scholar]
  31. Spitnik-Elson P., Elson D. Studies on the ribosome and its components. Prog Nucleic Acid Res Mol Biol. 1976;17:77–98. doi: 10.1016/s0079-6603(08)60066-5. [DOI] [PubMed] [Google Scholar]
  32. Spitnik-Elson P., Elson D. The fragmentation of ribosomes. Methods Enzymol. 1979;59:461–481. doi: 10.1016/0076-6879(79)59108-3. [DOI] [PubMed] [Google Scholar]
  33. Tal M. Thermal denaturation of ribosomes. Biochemistry. 1969 Jan;8(1):424–435. doi: 10.1021/bi00829a058. [DOI] [PubMed] [Google Scholar]
  34. Traub P., Nomura M. Structure and function of Escherichia coli ribosomes. VI. Mechanism of assembly of 30 s ribosomes studied in vitro. J Mol Biol. 1969 Mar 28;40(3):391–413. doi: 10.1016/0022-2836(69)90161-2. [DOI] [PubMed] [Google Scholar]
  35. Waring M. J. Complex formation between ethidium bromide and nucleic acids. J Mol Biol. 1965 Aug;13(1):269–282. doi: 10.1016/s0022-2836(65)80096-1. [DOI] [PubMed] [Google Scholar]
  36. Weiss R. L., Kimes B. W., Morris D. R. Cations and ribosome structure. 3. Effects on the 30S and 50S subunits of replacing bound Mg 2+ by inorganic cations. Biochemistry. 1973 Jan 30;12(3):450–456. doi: 10.1021/bi00727a014. [DOI] [PubMed] [Google Scholar]
  37. Zamir A., Miskin R., Elson D. Inactivation and reactivation of ribosomal subunits: amino acyl-transfer RNA binding activity of the 30 s subunit of Escherichia coli. J Mol Biol. 1971 Sep 14;60(2):347–364. doi: 10.1016/0022-2836(71)90299-3. [DOI] [PubMed] [Google Scholar]
  38. Zitomer R. S., Flaks J. G. Magnesium dependence and equilibrium of the Escherichia coli ribosomal subunit association. J Mol Biol. 1972 Nov 14;71(2):263–279. doi: 10.1016/0022-2836(72)90350-6. [DOI] [PubMed] [Google Scholar]

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