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. 1969 Nov;115(3):395–403. doi: 10.1042/bj1150395

An analysis of the ribosomal ribonucleic acids of Escherichia coli by hybridization techniques

R J Avery 1,2, J E M Midgley 1,2, G H Pigott 1,2
PMCID: PMC1185118  PMID: 4901070

Abstract

From analyses of the hybridization of Escherichia coli rRNA (ribosomal RNA) to homologous denatured DNA, the following conclusions were drawn. (1) When a fixed amount of DNA was hybridized with increasing amounts of RNA, only 0·35±0·02% of E. coli DNA was capable of binding (16s+23s) rRNA. Although preparations of 16s and 23s rRNA were virtually free from cross-contamination, the hybridization curves for purified 16s or 23s rRNA were almost identical with that of the parent specimen containing 1 weight unit of 16s rRNA mixed with 2 weight units of 23s rRNA. The 16s and 23s rRNA also competed effectively for the same specific DNA sites. It appears that these RNA species each possess all hybridizing species typical of the parent (16s+23s) rRNA specimen, though probably in different relative amounts. (2) By using hybridization-efficiency analysis of DNA–RNA hybridization curves (Avery & Midgley, 1969) it was found that (a) 0·45% of the DNA would hybridize total rRNA and (b) when so little RNA was added to unit weight of DNA that the DNA sites were not saturated, only 70–75% of the input RNA would form hybrids. The reasons for the discrepancy between the results obtained by the two alternative analytical approaches were discussed. (3) For either 16s or 23s rRNA, hybridization analysis indicated that two principal weight fractions of rRNA may exist, hybridizing to two distinct groups of DNA sites. However, these groups seem to be incompletely divided between the 16s and 23s fractions. Analysis suggested that (a) 85% of the 16s rRNA was hybridized to about half the DNA that specifically binds rRNA (0·23% of the total DNA). (b) 70% of the 23s rRNA hybridized to a further 0·23% of the DNA and (c) the minor fraction (15%) of 16s rRNA may be competitive with the major fraction (70%) of 23s rRNA. Conversely, the minor fraction (30%) of the 23s rRNA may compete with the major fraction (85%) of 16s rRNA. Models were proposed to explain the apparent lack of segregation of distinct RNA species in the two subfractions of rRNA. (4) If protein synthesis and ribosome maturation were inhibited in cells of an RCrel mutant, E. coli W 1665, by depriving them of an amino acid (methionine) essential for growth, the inhibition had no discernible effect on the relative rates of synthesis of rRNA species. The rRNA that accumulates in RCrel strains of E. coli after amino acid deprivation is apparently identical in its content of RNA species with that of the pre-existing mature RNA in the ribosomes. On the other hand, the messenger RNA is stabilized, and accumulates as about 15% of the RNA formed after withdrawal of the amino acid.

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Selected References

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

  1. ARONSON A. I., HOLOWCZYK M. A. COMPOSITION OF BACTERIAL RIBOSOMAL RNA. HETEROGENEITY WITHIN A GIVEN ORGANISM. Biochim Biophys Acta. 1965 Feb 8;95:217–231. doi: 10.1016/0005-2787(65)90487-9. [DOI] [PubMed] [Google Scholar]
  2. Attardi G., Huang P. C., Kabat S. Recognition of ribosomal RNA sites in DNA. I. Analysis of the E. coli system. Proc Natl Acad Sci U S A. 1965 Jun;53(6):1490–1498. doi: 10.1073/pnas.53.6.1490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BOREK E., RYAN A., ROCKENBACH J. Nucleic acid metabolism in relation to the lysogenic phenomenon. J Bacteriol. 1955 Apr;69(4):460–467. doi: 10.1128/jb.69.4.460-467.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DAGLEY S., TURNOCK G., WILD D. G. THE ACCUMULATION OF RIBONUCLEIC ACID BY A MUTANT OF ESCHERICHIA COLI. Biochem J. 1963 Sep;88:555–566. doi: 10.1042/bj0880555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. FLEISSNER E., BOREK E. A new enzyme of RNA synthesis: RNA methylase. Proc Natl Acad Sci U S A. 1962 Jul 15;48:1199–1203. doi: 10.1073/pnas.48.7.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fellner P., Sanger F. Sequence analysis of specific areas of the 16S and 23S ribosomal RNAs. Nature. 1968 Jul 20;219(5151):236–238. doi: 10.1038/219236a0. [DOI] [PubMed] [Google Scholar]
  7. Gillespie D., Spiegelman S. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J Mol Biol. 1965 Jul;12(3):829–842. doi: 10.1016/s0022-2836(65)80331-x. [DOI] [PubMed] [Google Scholar]
  8. Iwabuchi M., Kono M., Oumi T., Osawa S. The RNA components in ribonucleoprotein particles occurring during the course of ribosome formation in Escherichia coli. Biochim Biophys Acta. 1965 Oct 11;108(2):211–219. doi: 10.1016/0005-2787(65)90005-5. [DOI] [PubMed] [Google Scholar]
  9. KIT S. DEOXYRIBONUCLEIC ACIDS. Annu Rev Biochem. 1963;32:43–82. doi: 10.1146/annurev.bi.32.070163.000355. [DOI] [PubMed] [Google Scholar]
  10. LANE B. G. Studies of the terminal residues of high molecular weight ribonucleates. Can J Biochem Physiol. 1962 Aug;40:1071–1078. [PubMed] [Google Scholar]
  11. Lavallé R., De Hauwer G. Messenger RNA synthesis during amino acid starvation in Escherichia coli. J Mol Biol. 1968 Oct 28;37(2):269–288. doi: 10.1016/0022-2836(68)90267-2. [DOI] [PubMed] [Google Scholar]
  12. MANDELL J. D., HERSHEY A. D. A fractionating column for analysis of nucleic acids. Anal Biochem. 1960 Jun;1:66–77. doi: 10.1016/0003-2697(60)90020-8. [DOI] [PubMed] [Google Scholar]
  13. Maitra U., Hurwitz H. The role of DNA in RNA synthesis, IX. Nucleoside triphosphate termini in RNA polymerase products. Proc Natl Acad Sci U S A. 1965 Sep;54(3):815–822. doi: 10.1073/pnas.54.3.815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mangiarotti G., Apirion D., Schlessinger D., Silengo L. Biosynthetic precursors of 30S and 50S ribosomal particles in Escherichia coli. Biochemistry. 1968 Jan;7(1):456–472. doi: 10.1021/bi00841a058. [DOI] [PubMed] [Google Scholar]
  15. Midgley J. E., McIlreavy D. J. The chemical structure of bacterial ribosomal ribonucleic acid. II. Growth conditions and polynucleotide distribution in Escherichia coli ribosomal RNA. Biochim Biophys Acta. 1967 Jul 18;142(2):345–354. [PubMed] [Google Scholar]
  16. Midgley J. E. The estimation of polynucleotide chain length by a chemical method. Biochim Biophys Acta. 1965 Nov 8;108(3):340–347. doi: 10.1016/0005-2787(65)90026-2. [DOI] [PubMed] [Google Scholar]
  17. Moore R. L., McCarthy B. J. Comparative study of ribosomal ribonucleic acid cistrons in enterobacteria and myxobacteria. J Bacteriol. 1967 Oct;94(4):1066–1074. doi: 10.1128/jb.94.4.1066-1074.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nichols J. L., Lane B. G. The terminal groups of ribonucleate chains in each of the 16S and 23S components of Escherichia coli ribosomal RNA. Can J Biochem. 1967 Jun;45(6):937–948. doi: 10.1139/o67-104. [DOI] [PubMed] [Google Scholar]
  19. Oishi M., Sueoka N. Location of genetic loci of ribosomal RNA on Bacillus subtilis chromosome. Proc Natl Acad Sci U S A. 1965 Aug;54(2):483–491. doi: 10.1073/pnas.54.2.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pigott G. H., Midgley J. E. Characterization of rapidly labelled ribonucleic acid in Escherichia coli by deoxyribonucleic acid-ribonucleic acid hybridization. Biochem J. 1968 Nov;110(2):251–263. doi: 10.1042/bj1100251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Riley W. T. Polynucleotide chain lengths of rapidly labelled and ribosomal RNA of mammalian cells and E. coli. Nature. 1969 May 3;222(5192):446–452. doi: 10.1038/222446a0. [DOI] [PubMed] [Google Scholar]
  22. STENT G. S., BRENNER S. A genetic locus for the regulation of ribonucleic acid synthesis. Proc Natl Acad Sci U S A. 1961 Dec 15;47:2005–2014. doi: 10.1073/pnas.47.12.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schaup H. W., Best J. B., Goodman A. B. Evidence for heterogeneity of 23S RNA in E. coli. Nature. 1969 Mar 1;221(5183):864–865. doi: 10.1038/221864a0. [DOI] [PubMed] [Google Scholar]
  24. Smith I., Dubnau D., Morrell P., Marmur J. Chromosomal location of DNA base sequences complementary to transfer RNA and to 5 s, 16 s and 23 s ribosomal RNA in Bacillus subtilis. J Mol Biol. 1968 Apr 14;33(1):123–140. doi: 10.1016/0022-2836(68)90285-4. [DOI] [PubMed] [Google Scholar]
  25. Stanley W. M., Jr, Bock R. M. Isolation and physical properties of the ribosomal ribonucleic acid of Escherichia coli. Biochemistry. 1965 Jul;4(7):1302–1311. doi: 10.1021/bi00883a014. [DOI] [PubMed] [Google Scholar]
  26. Stubbs J. D., Hall B. D. Effects of amino acid starvation upon constitutive tryptophan messenger RNA synthesis. J Mol Biol. 1968 Oct 28;37(2):303–312. doi: 10.1016/0022-2836(68)90269-6. [DOI] [PubMed] [Google Scholar]
  27. Walker G. J. Metabolism of the reserve polysaccharide of Streptococcus mitis. Some properties of a pullulanase. Biochem J. 1968 Jun;108(1):33–40. doi: 10.1042/bj1080033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. YANKOFSKY S. A., SPIEGELMAN S. Distinct cistrons for the two ribosomal RNA components. Proc Natl Acad Sci U S A. 1963 Apr;49:538–544. doi: 10.1073/pnas.49.4.538. [DOI] [PMC free article] [PubMed] [Google Scholar]

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