Specific Cross-linking of Proteins S7 and L4 to Ribosomal RNA, by UV Irradiation of Escherichia Coli Ribosomal Subunits

Overview

Journal Mol Gen Genet

Specialties Genetics
Molecular Biology

Date 1975 Dec 9

PMID 814400

Citations 32

Authors

K Moller

R Brimacombe

Affiliations

Soon will be listed here.

Abstract

Radioactive 30S and 50S subunits from E. coli ribosomes were irradiated with UV light, under conditions giving rise to approximately 10% cross-linking of protein to ribosomal RNA. Irradiation to levels of cross-linking higher than 10% caused unfolding of the ribosomal subunits, even in the presence of 5 mM magnesium. The specificity of the cross-linking reaction at this low level was found to be extremely high. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease and then analysed on Sarkosyl gels. S7 was found to be the primary target of the cross-linking reaction in the 30S particle. This was proven by using subunits from both E. coli MRE 600 and A19, whose respective S7 species differ markedly. In the 50S particle, L4 was the primary target, although L2 was also cross-linked to a small extent. Ambiguity in the identification of L4 in the Sarkosyl system was resolved by two-dimensional electrophoresis, which was also used to demonstrate a genuine linkage to RNA in the case of both S7 and L4; proteins spots containing 32P were observed, derived from these two proteins, when subunits containing 32P-RNA were irradiated, treated with nuclease, and applied to the electrophoresis. The identities of S7, L4 and L2 were finally confirmed by Ouchterlony tests with protein-specific anti-sera.

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References

Spierer P, Zimmerman R, Mackie G . RNA-protein interactions in the ribosome. Binding of 50-S-subunit proteins to 5' and 3' terminal segments of the 23-S RNA. Eur J Biochem. 1975; 52(3):459-68. DOI: 10.1111/j.1432-1033.1975.tb04014.x. View

Stoffler G, WITTMANN H . Sequence differences of Escherichia coli 30S ribosomal proteins as determined by immunochemical methods. Proc Natl Acad Sci U S A. 1971; 68(9):2283-7. PMC: 389401. DOI: 10.1073/pnas.68.9.2283. View

Morgan J, Brimacombe R . A series of specific ribonucleoprotein fragments from the 30-S subparticle of Escherichia coli ribosomes. Eur J Biochem. 1972; 29(3):542-52. DOI: 10.1111/j.1432-1033.1972.tb02020.x. View

Markovitz A . Ultraviolet light-induced stable complexes of DNA and DNA polymerase. Biochim Biophys Acta. 1972; 281(4):522-34. DOI: 10.1016/0005-2787(72)90153-0. View

Zimmermann R, Mackie G, Muto A, Garrett R, Ungewickell E, Ehresmann C . Location and characteristics of ribosomal protein binding sites in the 16S RNA of Escherichia coli. Nucleic Acids Res. 1975; 2(2):279-302. PMC: 342832. DOI: 10.1093/nar/2.2.279. View

Newton I, Rinke J, Brimacombe R . Random exchange of ribosomal proteins in EDTA sub-particles. FEBS Lett. 1975; 51(1):215-8. DOI: 10.1016/0014-5793(75)80890-8. View

Dahlberg A, DINGMAN C, Peacock A . Electrophoretic characterization of bacterial polyribosomes in agarose-acrylamide composite gels. J Mol Biol. 1969; 41(1):139-47. DOI: 10.1016/0022-2836(69)90131-4. View

Barritault D, Milet M, Hayes D . Identification of neighbouring proteins in the 30 S ribosomes of E. coli. FEBS Lett. 1975; 50(2):114-20. DOI: 10.1016/0014-5793(75)80469-8. View

Brimacombe R, Morgan J, Oakley D, Cox R . Specific ribonucleoprotein fragment from the 30S subunit of E. coli ribosomes. Nat New Biol. 1971; 231(24):209-12. DOI: 10.1038/newbio231209a0. View

10.

Branlant C, Krol A, Sriwidada J, Ebel J, Sloof P, Garrett R . A partial localisation of the binding sites of the 50 S subunit proteins L1, L20 and L23 on 23 S ribosomal RNA of Escherichia coli. FEBS Lett. 1975; 52(2):195-201. DOI: 10.1016/0014-5793(75)80805-2. View

11.

Yuki A, Brimacombe R . Nucleotide sequences of Escherichia coli 16-S RNA associated with ribosomal proteins S7, S9, S10, S14 and S19. Eur J Biochem. 1975; 56(1):23-34. DOI: 10.1111/j.1432-1033.1975.tb02203.x. View

12.

Newton I, Brimacombe R . Two specific ribonucleoprotein fragments from the 50-S sub-particle of Escherichia coli ribosomes. Eur J Biochem. 1974; 48(2):513-8. DOI: 10.1111/j.1432-1033.1974.tb03792.x. View

13.

ANKER H . A solubilizable acrylamide gel for electrophoresis. FEBS Lett. 1970; 7(3):293. DOI: 10.1016/0014-5793(70)80185-5. View

14.

Morgan J, Brimacombe R . A preliminary three-dimensional arrangement of the proteins in the Escherichia coli 30-S ribosomal sub-particle. Eur J Biochem. 1973; 37(3):472-80. DOI: 10.1111/j.1432-1033.1973.tb03008.x. View

15.

Schenkman M, Ward D, Moore P . Covalent attachment of a messenger RNA to the Escherichia coli ribosome. Biochim Biophys Acta. 1974; 353(4):503-8. DOI: 10.1016/0005-2787(74)90056-2. View

16.

Gorelic L . Photoinduced convalent crosslinkage, in situ, of Escherichia coli 50 S ribosomal proteins to rRNA. Biochim Biophys Acta. 1975; 390(2):209-25. DOI: 10.1016/0005-2787(75)90342-1. View

17.

Kaltschmidt E, WITTMANN H . Ribosomal proteins. VII. Two-dimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins. Anal Biochem. 1970; 36(2):401-12. DOI: 10.1016/0003-2697(70)90376-3. View

18.

Kaltschmidt E, Stoffler G, Dzionara M, WITTMANN H . Ribosomal proteins. XVII. Comparative studies on ribosomal proteins of four strains of Escherichia coli. Mol Gen Genet. 1970; 109(4):303-8. DOI: 10.1007/BF00267700. View

19.

Schoemaker H, Schimmel P . Photo-induced joining of a transfer RNA with its cognate aminoacyl-transfer RNA synthetase. J Mol Biol. 1974; 84(4):503-13. DOI: 10.1016/0022-2836(74)90112-0. View