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PMID: 6364140 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Nuclease protection analysis of ribonucleoprotein complexes: use of the cytotoxic ribonuclease alpha-sarcin to determine the binding sites for Escherichia coli ribosomal proteins L5, L18, and L25 on 5S rRNA.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 81 ·No. 2 ·1984-01-00 ·Pages 322-6

Huber PW, Wool IG

Abstract

A rapid and convenient method has been devised to determine the binding sites for proteins on RNA. The procedure is an adaptation of one used to map DNA-protein complexes by protection against nuclease digestion. The method uses the cytotoxic ribonuclease alpha-sarcin, which hydrolyzes purines in both single- and double-stranded regions of RNA. It has been authenticated by confirming the binding sites for the Escherichia coli ribosomal proteins L18 and L25 on 5S rRNA and its value has been established by identifying the attachment site for protein L5. The procedure should be useful for the analysis of other ribonucleoprotein complexes.

MeSH Terms
Binding Sites Endoribonucleases Escherichia coli/ultrastructure Fungal Proteins Nucleic Acid Conformation RNA, Ribosomal/metabolism Ribonucleases Ribonucleoproteins Ribosomal Proteins/metabolism Ribosomes/ultrastructure
Chemicals
Fungal Proteins RNA, Ribosomal Ribonucleoproteins Ribosomal Proteins alpha-sarcin Endoribonucleases Ribonucleases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Huber P W
Wool I G
References (30)
30 references, click to expand
  1. Isolation and characterization of 5S RNA-protein complexes from Bacillus stearothermophilus and Escherichia coli ribosomes.
    Mol Gen Genet. 1972;119(4):337-44 PMID: 4567807
  2. ALPHA SARCIN, A NEW ANTITUMOR AGENT. II. FERMENTATION AND ANTITUMOR SPECTRUM.
    Appl Microbiol. 1965 May;13:322-6 PMID: 14330325
  3. The sequence of steps in the attachment of 5-S RNA to cores of Escherichia coli ribosomes.
    Biochim Biophys Acta. 1973 Oct 26;324(3):375-85 PMID: 4586940
  4. Identification of the nucleotide sequences involved in the interaction between Escherichia coli 5 RNA and specific 50 S subunit proteins.
    J Mol Biol. 1973 Jun 15;77(1):133-52 PMID: 4588880
  5. Affinity binding of Escherichia coli ribosomal proteins to immobilized RNA.
    FEBS Lett. 1975 Jan 1;49(3):306-9 PMID: 1089065
  6. 5S RNA secondary structure.
    Nature. 1975 Aug 7;256(5517):505-7 PMID: 808733
  7. Specific cleavage of ribosomal RNA caused by alpha sarcin.
    Nucleic Acids Res. 1977 Apr;4(4):1097-1110 PMID: 325525
  8. Effects of some proteins that inactivate the eukaryotic ribosome.
    FEBS Lett. 1977;78(1):143-6 PMID: 872934
  9. Mapping adenines, guanines, and pyrimidines in RNA.
    Nucleic Acids Res. 1977 Aug;4(8):2527-38 PMID: 409999
  10. The mode of action of alpha sarcin and a novel assay of the puromycin reaction.
    Biochem J. 1978 Jan 15;170(1):57-61 PMID: 629783
  11. Identification of Escherichia coli and Bacillus stearothermophilus ribosomal protein binding sites on Escherichia coli 5S RNA.
    Mol Gen Genet. 1978 Apr 17;160(3):247-57 PMID: 353489
  12. Stoichiometry, cooperativity, and stability of interactions between 5S RNA and proteins L5, L18, and L25 from the 50S ribosomal subunit of Escherichia coli.
    Biochemistry. 1978 Jun 27;17(13):2474-9 PMID: 354687
  13. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity.
    Nucleic Acids Res. 1978 Sep;5(9):3157-70 PMID: 212715
  14. Reactions at the termini of tRNA with T4 RNA ligase.
    Nucleic Acids Res. 1978 Oct;5(10):3665-77 PMID: 724498
  15. Parameters for the interaction of ribosomal proteins L5, L18, and L25 with 5S RNA from Escherichia coli.
    Biochemistry. 1978 Dec 12;17(25):5394-8 PMID: 365228
  16. Use of in vitro 32P labeling in the sequence analysis of nonradioactive tRNAs.
    Methods Enzymol. 1979;59:58-109 PMID: 220499
  17. Cooperative interactions among protein and RNA components of the 50S ribosomal subunit of Escherichia coli.
    Nucleic Acids Res. 1979 Apr;6(4):1669-82 PMID: 109811
  18. Direct chemical method for sequencing RNA.
    Proc Natl Acad Sci U S A. 1979 Apr;76(4):1760-4 PMID: 377283
  19. A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25.
    Nucleic Acids Res. 1979 Jun 11;6(7):2453-70 PMID: 379819
  20. Structure of 5 S ribosomal RNA from Escherichia coli: identification of kethoxal-reactive sites in the A and B conformations.
    J Mol Biol. 1979 Aug 25;132(4):621-36 PMID: 393828
  21. Structures of complexes of 5S RNA with ribosomal proteins L5, L18 and L25 from Escherichia coli: identification of kethoxal-reactive sites on the 5S RNA.
    J Mol Biol. 1979 Aug 25;132(4):637-48 PMID: 393829
  22. Structural analyses of E. coli 5S RNA fragments, their associates and complexes with proteins L18 and L25.
    Nucleic Acids Res. 1982 Feb 11;10(3):947-65 PMID: 6278442
  23. A "bulged" double helix in a RNA-protein contact site.
    Proc Natl Acad Sci U S A. 1981 Dec;78(12):7331-5 PMID: 7038676
  24. Binding site of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases.
    Biochemistry. 1982 May 11;21(10):2313-20 PMID: 6178424
  25. The site of action of alpha-sarcin on eukaryotic ribosomes. The sequence at the alpha-sarcin cleavage site in 28 S ribosomal ribonucleic acid.
    J Biol Chem. 1982 Aug 10;257(15):9054-60 PMID: 7047533
  26. Three-dimensional structural model of eubacterial 5S RNA that has functional implications.
    Proc Natl Acad Sci U S A. 1982 Aug;79(15):4599-603 PMID: 6181508
  27. The ribonuclease activity of the cytotoxin alpha-sarcin. The characteristics of the enzymatic activity of alpha-sarcin with ribosomes and ribonucleic acids as substrates.
    J Biol Chem. 1983 Feb 25;258(4):2662-7 PMID: 6185500
  28. Nuclear Overhauser experiments at 500 MHz on the downfield proton spectra of 5S ribonucleic acid and its complex with ribosomal protein L25.
    Biochemistry. 1983 May 24;22(11):2622-9 PMID: 6347250
  29. ALPHA SARCIN, A NEW ANTITUMOR AGENT. I. ISOLATION, PURIFICATION, CHEMICAL COMPOSITION, AND THE IDENTITY OF A NEW AMINO ACID.
    Appl Microbiol. 1965 May;13:314-21 PMID: 14325268
  30. Selective reaction of glyoxal with guanine residues in native and denatured Escherichia coli 5S RNA.
    Biochimie. 1973;55(2):135-42 PMID: 4198187
Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.81.2.322
Published
1984-01-00
Pages
322-6
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC344668
Subset
IM
Grants
NCI NIH HHS · CA 19265 · United States
NIGMS NIH HHS · GM 21769 · United States
External Links
PubMed source DOI: 10.1073/pnas.81.2.322 PMC Full Text View journal details
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