Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1985 Apr;4(4):1019–1024. doi: 10.1002/j.1460-2075.1985.tb03733.x

Does unpaired adenosine-66 from helix II of Escherichia coli 5S RNA bind to protein L18?

J Christiansen, S R Douthwaite, A Christensen, R A Garrett
PMCID: PMC554294  PMID: 2990903

Abstract

Adenosine-66 is unpaired within helix II of Escherichia coli 5S RNA and lies in the binding site of ribosomal protein L18. It has been proposed as a recognition site for protein L18. We have investigated further the structural importance of this nucleotide by deleting it. The 5S RNA gene of the rrnB operon of E. coli was subjected to primer-directed mutagenesis. To produce the deletion it was necessary to use simultaneously the mutagenic dodecamer dCGGCGCACGGCG and the universal M13 primer dCCCAGTCACGACGTT, and to employ forced annealing conditions. The mutated gene was expressed in an overproducing plasmid derived from pKK3535. Binding studies with protein L18 revealed that the protein bound much more weakly to the mutated 5S RNA. We consider the most likely explanation of this result is that L18 interacts with adenosine-66, and we present a tentative model for an interaction between the unpaired adenosine and the adjacent guanosine-67 of the RNA and glutamine-19 of the protein L18.

Full text

PDF
1019

Images in this article

1021Fig. 4.
on p.1021
1021Fig. 5.
on p.1021
1021Fig. 6.
on p.1021

Selected References

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

  1. Brosius J., Dull T. J., Sleeter D. D., Noller H. F. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol. 1981 May 15;148(2):107–127. doi: 10.1016/0022-2836(81)90508-8. [DOI] [PubMed] [Google Scholar]
  2. Douthwaite S., Christensen A., Garrett R. A. Binding site of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases. Biochemistry. 1982 May 11;21(10):2313–2320. doi: 10.1021/bi00539a007. [DOI] [PubMed] [Google Scholar]
  3. Fuenteun J., Monier R., Garrett R., Le Bret M., Le Pecq J. B. Effect of 50 S subunit proteins L5, L18 and L25 on the fluorescence of 5 S RNA-bound ethidium bromide. J Mol Biol. 1975 Apr 25;93(4):535–541. doi: 10.1016/0022-2836(75)90245-4. [DOI] [PubMed] [Google Scholar]
  4. Gillam S., Smith M. Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: I. Optimum conditions and minimum ologodeoxyribonucleotide length. Gene. 1979 Dec;8(1):81–97. doi: 10.1016/0378-1119(79)90009-x. [DOI] [PubMed] [Google Scholar]
  5. Glickman B. W. Spontaneous mutagenesis in Escherichia coli strains lacking 6-methyladenine residues in their DNA: an altered mutational spectrum in dam- mutants. Mutat Res. 1979 Jul;61(2):153–162. doi: 10.1016/0027-5107(79)90122-2. [DOI] [PubMed] [Google Scholar]
  6. Göringer H. U., Wagner R., Jacob W. F., Dahlberg A. E., Zwieb C. Oligonucleotide directed mutagenesis of Escherichia coli 5S ribosomal RNA: construction of mutant and structural analysis. Nucleic Acids Res. 1984 Sep 25;12(18):6935–6950. doi: 10.1093/nar/12.18.6935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Herrmann R., Neugebauer K., Pirkl E., Zentgraf H., Schaller H. Conversion of bacteriophage fd into an efficient single-stranded DNA vector system. Mol Gen Genet. 1980 Jan;177(2):231–242. doi: 10.1007/BF00267434. [DOI] [PubMed] [Google Scholar]
  8. Hindennach I., Kaltschmidt E., Wittmann H. G. Ribosomal proteins. Isolation of proteins from 50S ribosomal subunits of Escherichia coli. Eur J Biochem. 1971 Nov 11;23(1):12–16. doi: 10.1111/j.1432-1033.1971.tb01585.x. [DOI] [PubMed] [Google Scholar]
  9. Jinks-Robertson S., Gourse R. L., Nomura M. Expression of rRNA and tRNA genes in Escherichia coli: evidence for feedback regulation by products of rRNA operons. Cell. 1983 Jul;33(3):865–876. doi: 10.1016/0092-8674(83)90029-6. [DOI] [PubMed] [Google Scholar]
  10. Jordan B. R., Forget B. G., Monier R. A low molecular weight ribonucleic acid synthesized by Escherichia coli in the presence of chloramphenicol: characterization and relation to normally synthesized 5 s ribonucleic acid. J Mol Biol. 1971 Feb 14;55(3):407–421. doi: 10.1016/0022-2836(71)90326-3. [DOI] [PubMed] [Google Scholar]
  11. Kramer B., Kramer W., Fritz H. J. Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli. Cell. 1984 Oct;38(3):879–887. doi: 10.1016/0092-8674(84)90283-6. [DOI] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Marinus M. G. Location of DNA methylation genes on the Escherichia coli K-12 genetic map. Mol Gen Genet. 1973 Dec 14;127(1):47–55. doi: 10.1007/BF00267782. [DOI] [PubMed] [Google Scholar]
  14. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  16. Newberry V., Brosius J., Garrett R. Fragment of protein L18 from the Escherichia coli ribosome that contains the 5S RNA binding site. Nucleic Acids Res. 1978 Jun;5(6):1753–1766. doi: 10.1093/nar/5.6.1753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Norris K., Norris F., Christiansen L., Fiil N. Efficient site-directed mutagenesis by simultaneous use of two primers. Nucleic Acids Res. 1983 Aug 11;11(15):5103–5112. doi: 10.1093/nar/11.15.5103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Osterberg R., Garrett R. A. Small-angle X-ray titration study on the complex formation between 5-S RNA and the L18 protein of the Escherichia coli 50-S ribosome particle. Eur J Biochem. 1977 Sep 15;79(1):56–72. doi: 10.1111/j.1432-1033.1977.tb11784.x. [DOI] [PubMed] [Google Scholar]
  20. Peattie D. A. Direct chemical method for sequencing RNA. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1760–1764. doi: 10.1073/pnas.76.4.1760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Peattie D. A., Douthwaite S., Garrett R. A., Noller H. F. A "bulged" double helix in a RNA-protein contact site. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7331–7335. doi: 10.1073/pnas.78.12.7331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  23. Southern E. Gel electrophoresis of restriction fragments. Methods Enzymol. 1979;68:152–176. doi: 10.1016/0076-6879(79)68011-4. [DOI] [PubMed] [Google Scholar]
  24. Spierer P., Bogdanov A. A., Zimmermann R. A. Parameters for the interaction of ribosomal proteins L5, L18, and L25 with 5S RNA from Escherichia coli. Biochemistry. 1978 Dec 12;17(25):5394–5398. doi: 10.1021/bi00618a012. [DOI] [PubMed] [Google Scholar]
  25. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. van Wezenbeek P. M., Hulsebos T. J., Schoenmakers J. G. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd. Gene. 1980 Oct;11(1-2):129–148. doi: 10.1016/0378-1119(80)90093-1. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES