Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1975 Apr;72(4):1505–1509. doi: 10.1073/pnas.72.4.1505

Affinity labeling of the ribosomal decoding site with an AUG-substrate analog.

O Pongs, E Lanka
PMCID: PMC432565  PMID: 1093183

Abstract

The trinucleotide AUG was condensed at the 5'-end with N-bromoacetyl-p-aminophenylphosphate. This bromoactylated AUG analog reacted irreversibly with the mRNA binding site of Escherichia coli 70S ribosomes. After reaction of 70S ribosomes with the AUG analog, labeled 30S subunits could be isolated that were programmed for initiation-factor-dependent binding of fMet-tRNAfMet. This shows that this AUG-affinity label reacted in the decoding site for fMet-tRNAfMet. By combination of sodium dodecyl sulfate-, Sarkosyl-, and ureapolyacrylamide gel electrophoresis the AUG-affinity label was found to be irreversibly bound to ribosomal proteins S4, the ram gene product, and S18.

Full text

PDF
1505

Images in this article

1507Image
on p.1507
1508Image
on p.1508
1508Image
on p.1508

Selected References

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

  1. Brimacombe R., Morgan J. M., Cox R. A. An improved technique for the analysis of ribonucleoprotein fragments from Escherichia coli 30S ribosomes. Eur J Biochem. 1971 Nov 11;23(1):52–60. doi: 10.1111/j.1432-1033.1971.tb01591.x. [DOI] [PubMed] [Google Scholar]
  2. Cuatrecasas P., Wilchek M., Anfinsen C. B. Affinity labeling of the active site of staphylococcal nuclease. Reactions with bromoacetylated substrate analogues. J Biol Chem. 1969 Aug 25;244(16):4316–4329. [PubMed] [Google Scholar]
  3. Czernilofsky A. P., Collatz E. E., Stöffler G., Kuechler E. Proteins at the tRNA binding sites of Escherichia coli ribosomes. Proc Natl Acad Sci U S A. 1974 Jan;71(1):230–234. doi: 10.1073/pnas.71.1.230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dzionara M., Kaltschmidt E., Wittmann H. G. Ribosomal proteins. 8. Molecular weights of isolated ribosomal proteins of Escherichia coli. Proc Natl Acad Sci U S A. 1970 Dec;67(4):1909–1913. doi: 10.1073/pnas.67.4.1909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Erdmann V. A., Fahnestock S., Higo K., Nomura M. Role of 5S RNA in the functions of 50S ribosomal subunits. Proc Natl Acad Sci U S A. 1971 Dec;68(12):2932–2936. doi: 10.1073/pnas.68.12.2932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gorini L. Ribosomal discrimination of tRNAs. Nat New Biol. 1971 Dec 29;234(52):261–264. doi: 10.1038/newbio234261a0. [DOI] [PubMed] [Google Scholar]
  7. Hardy S. J., Kurland C. G. The relationship between poly A polymerase and the ribosomes. Biochemistry. 1966 Nov;5(11):3676–3684. doi: 10.1021/bi00875a042. [DOI] [PubMed] [Google Scholar]
  8. Hasenbank R., Guthrie C., Stöffler G., Wittmann H. G., Rosen L., Apirion D. Electrophoretic and immunological studies on ribosomal proteins of 100 Escherichia coli revertants from streptomycin dependence. Mol Gen Genet. 1973 Dec 14;127(1):1–18. doi: 10.1007/BF00267778. [DOI] [PubMed] [Google Scholar]
  9. Held W. A., Gette W. R., Nomura M. Role of 16S ribosomal ribonucleic acid and the 30S ribosomal protein S12 in the initiation of natural messenger ribonucleic acid translation. Biochemistry. 1974 May 7;13(10):2115–2122. doi: 10.1021/bi00707a019. [DOI] [PubMed] [Google Scholar]
  10. Hershey J. W., Thach R. E. Role of guanosine 5'-triphosphate in the initiation of Peptide synthesis, I. Synthesis of formylmethionyl-puromycin. Proc Natl Acad Sci U S A. 1967 Mar;57(3):759–766. doi: 10.1073/pnas.57.3.759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Highland J. H., Ochsner E., Gordon J., Hasenbank R., Stöffler G. Letter: Inhibition of phenylalanyl-tRNA binding and elongation factor Tu-dependent GTP hydrolysis by antibodies specific for several ribosomal proteins. J Mol Biol. 1974 Jun 15;86(1):175–178. doi: 10.1016/s0022-2836(74)80017-3. [DOI] [PubMed] [Google Scholar]
  12. Kaltschmidt E., Wittmann H. G. Ribosomal proteins. XII. Number of proteins in small and large ribosomal subunits of Escherichia coli as determined by two-dimensional gel electrophoresis. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1276–1282. doi: 10.1073/pnas.67.3.1276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Muto A., Ehresmann C., Fellner P., Zimmermann R. A. RNA-protein interactions in the ribosome. I. Characterization and ribonuclease digestion of 16 S RNA-ribosomal protein complexes. J Mol Biol. 1974 Jun 25;86(2):411–432. doi: 10.1016/0022-2836(74)90028-x. [DOI] [PubMed] [Google Scholar]
  14. NIRENBERG M. W., MATTHAEI J. H. The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci U S A. 1961 Oct 15;47:1588–1602. doi: 10.1073/pnas.47.10.1588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nomura M. Assembly of bacterial ribosomes. Science. 1973 Mar 2;179(4076):864–873. doi: 10.1126/science.179.4076.864. [DOI] [PubMed] [Google Scholar]
  16. Nomura M., Mizushima S., Ozaki M., Traub P., Lowry C. V. Structure and function of ribosomes and their molecular components. Cold Spring Harb Symp Quant Biol. 1969;34:49–61. doi: 10.1101/sqb.1969.034.01.009. [DOI] [PubMed] [Google Scholar]
  17. Oen H., Pellegrini M., Eilat D., Cantor C. R. Identification of 50S proteins at the peptidyl-tRNA binding site of Escherichia coli ribosomes. Proc Natl Acad Sci U S A. 1973 Oct;70(10):2799–2803. doi: 10.1073/pnas.70.10.2799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pongs O., Bald R., Erdmann V. A. Identification of chloramphenicol-binding protein in Escherichia coli ribosomes by affinity labeling. Proc Natl Acad Sci U S A. 1973 Aug;70(8):2229–2233. doi: 10.1073/pnas.70.8.2229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. RAZZELL W. E., KHORANA H. G. Studies on polynucleotides. III. Enzymic degradation; substrate specificity and properties of snake venom phosphodiesterase. J Biol Chem. 1959 Aug;234(8):2105–2113. [PubMed] [Google Scholar]
  20. Studier F. W. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol Biol. 1973 Sep 15;79(2):237–248. doi: 10.1016/0022-2836(73)90003-x. [DOI] [PubMed] [Google Scholar]
  21. Zimmermann R. A., Garvin R. T., Gorini L. Alteration of a 30S ribosomal protein accompanying the ram mutation in Escherichia coli. Proc Natl Acad Sci U S A. 1971 Sep;68(9):2263–2267. doi: 10.1073/pnas.68.9.2263. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES