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. 2001 Apr;7(4):598–609. doi: 10.1017/s1355838201002059

A short fragment of 23S rRNA containing the binding sites for two ribosomal proteins, L24 and L4, is a key element for rRNA folding during early assembly.

U Stelzl 1, K H Nierhaus 1
PMCID: PMC1370113  PMID: 11345438

Abstract

Previously we described an in vitro selection variant abbreviated SERF (in vitro selection from random rRNA fragments) that identifies protein binding sites within large RNAs. With this method, a small rRNA fragment derived from the 23S rRNA was isolated that binds simultaneously and independently the ribosomal proteins L4 and L24 from Escherichia coli. Until now the rRNA structure within the ternary complex L24-rRNA-L4 could not be studied due to the lack of an appropriate experimental strategy. Here we tackle the issue by separating the various complexes via native gel-electrophoresis and analyzing the rRNA structure by in-gel iodine cleavage of phosphorothioated RNA. The results demonstrate that during the transition from either the L4 or L24 binary complex to the ternary complex the structure of the rRNA fragment changes significantly. The identified protein binding sites are in excellent agreement with the recently reported crystal structure of the 50S subunit. Because both proteins play a prominent role in early assembly of the large subunit, the results suggest that the identified rRNA fragment is a key element for the folding of the 23S RNA during early assembly. The introduced in-gel cleavage method should be useful when an RNA structure within mixed populations of different but related complexes should be studied.

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Selected References

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  1. Ban N., Nissen P., Hansen J., Moore P. B., Steitz T. A. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science. 2000 Aug 11;289(5481):905–920. doi: 10.1126/science.289.5481.905. [DOI] [PubMed] [Google Scholar]
  2. Cate J. H., Hanna R. L., Doudna J. A. A magnesium ion core at the heart of a ribozyme domain. Nat Struct Biol. 1997 Jul;4(7):553–558. doi: 10.1038/nsb0797-553. [DOI] [PubMed] [Google Scholar]
  3. Dabrowski M., Spahn C. M., Nierhaus K. H. Interaction of tRNAs with the ribosome at the A and P sites. EMBO J. 1995 Oct 2;14(19):4872–4882. doi: 10.1002/j.1460-2075.1995.tb00168.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dabrowski M., Spahn C. M., Schäfer M. A., Patzke S., Nierhaus K. H. Protection patterns of tRNAs do not change during ribosomal translocation. J Biol Chem. 1998 Dec 4;273(49):32793–32800. doi: 10.1074/jbc.273.49.32793. [DOI] [PubMed] [Google Scholar]
  5. Dertinger D., Behlen L. S., Uhlenbeck O. C. Using phosphorothioate-substituted RNA to investigate the thermodynamic role of phosphates in a sequence specific RNA-protein complex. Biochemistry. 2000 Jan 11;39(1):55–63. doi: 10.1021/bi991769v. [DOI] [PubMed] [Google Scholar]
  6. Diedrich G., Burkhardt N., Nierhaus K. H. Large-scale isolation of proteins of the large subunit from Escherichia coli ribosomes. Protein Expr Purif. 1997 Jun;10(1):42–50. doi: 10.1006/prep.1996.0702. [DOI] [PubMed] [Google Scholar]
  7. Dohme F., Nierhaus K. H. Total reconstitution and assembly of 50 S subunits from Escherichia coli Ribosomes in vitro. J Mol Biol. 1976 Nov 15;107(4):585–599. doi: 10.1016/s0022-2836(76)80085-x. [DOI] [PubMed] [Google Scholar]
  8. Draper D. E., Deckman I. C., Vartikar J. V. Physical studies of ribosomal protein-RNA interactions. Methods Enzymol. 1988;164:203–220. doi: 10.1016/s0076-6879(88)64044-4. [DOI] [PubMed] [Google Scholar]
  9. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  10. Egebjerg J., Leffers H., Christensen A., Andersen H., Garrett R. A. Structure and accessibility of domain I of Escherichia coli 23 S RNA in free RNA, in the L24-RNA complex and in 50 S subunits. Implications for ribosomal assembly. J Mol Biol. 1987 Jul 5;196(1):125–136. doi: 10.1016/0022-2836(87)90515-8. [DOI] [PubMed] [Google Scholar]
  11. Gish G., Eckstein F. DNA and RNA sequence determination based on phosphorothioate chemistry. Science. 1988 Jun 10;240(4858):1520–1522. doi: 10.1126/science.2453926. [DOI] [PubMed] [Google Scholar]
  12. Gregory S. T., Dahlberg A. E. Erythromycin resistance mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23 S ribosomal RNA. J Mol Biol. 1999 Jun 18;289(4):827–834. doi: 10.1006/jmbi.1999.2839. [DOI] [PubMed] [Google Scholar]
  13. Liiv A., Tenson T., Remme J. Analysis of the ribosome large subunit assembly and 23 S rRNA stability in vivo. J Mol Biol. 1996 Nov 1;263(3):396–410. doi: 10.1006/jmbi.1996.0584. [DOI] [PubMed] [Google Scholar]
  14. Malhotra A., Penczek P., Agrawal R. K., Gabashvili I. S., Grassucci R. A., Jünemann R., Burkhardt N., Nierhaus K. H., Frank J. Escherichia coli 70 S ribosome at 15 A resolution by cryo-electron microscopy: localization of fMet-tRNAfMet and fitting of L1 protein. J Mol Biol. 1998 Jul 3;280(1):103–116. doi: 10.1006/jmbi.1998.1859. [DOI] [PubMed] [Google Scholar]
  15. Milligan J. F., Uhlenbeck O. C. Determination of RNA-protein contacts using thiophosphate substitutions. Biochemistry. 1989 Apr 4;28(7):2849–2855. doi: 10.1021/bi00433a016. [DOI] [PubMed] [Google Scholar]
  16. Nierhaus K. H. The assembly of prokaryotic ribosomes. Biochimie. 1991 Jun;73(6):739–755. doi: 10.1016/0300-9084(91)90054-5. [DOI] [PubMed] [Google Scholar]
  17. Nowotny V., Nierhaus K. H. Initiator proteins for the assembly of the 50S subunit from Escherichia coli ribosomes. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7238–7242. doi: 10.1073/pnas.79.23.7238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Powers T., Daubresse G., Noller H. F. Dynamics of in vitro assembly of 16 S rRNA into 30 S ribosomal subunits. J Mol Biol. 1993 Jul 20;232(2):362–374. doi: 10.1006/jmbi.1993.1396. [DOI] [PubMed] [Google Scholar]
  19. Powers T., Noller H. F. Hydroxyl radical footprinting of ribosomal proteins on 16S rRNA. RNA. 1995 Apr;1(2):194–209. [PMC free article] [PubMed] [Google Scholar]
  20. Revzin A., Ceglarek J. A., Garner M. M. Comparison of nucleic acid-protein interactions in solution and in polyacrylamide gels. Anal Biochem. 1986 Feb 15;153(1):172–177. doi: 10.1016/0003-2697(86)90077-1. [DOI] [PubMed] [Google Scholar]
  21. Rudinger J., Puglisi J. D., Pütz J., Schatz D., Eckstein F., Florentz C., Giegé R. Determinant nucleotides of yeast tRNA(Asp) interact directly with aspartyl-tRNA synthetase. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5882–5886. doi: 10.1073/pnas.89.13.5882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schatz D., Leberman R., Eckstein F. Interaction of Escherichia coli tRNA(Ser) with its cognate aminoacyl-tRNA synthetase as determined by footprinting with phosphorothioate-containing tRNA transcripts. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6132–6136. doi: 10.1073/pnas.88.14.6132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Skinner R. H., Stark M. J., Dahlberg A. E. Mutations within the 23S rRNA coding sequence of E. coli which block ribosome assembly. EMBO J. 1985 Jun;4(6):1605–1608. doi: 10.1002/j.1460-2075.1985.tb03824.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Smith J. S., Nikonowicz E. P. Phosphorothioate substitution can substantially alter RNA conformation. Biochemistry. 2000 May 16;39(19):5642–5652. doi: 10.1021/bi992712b. [DOI] [PubMed] [Google Scholar]
  25. Spillmann S., Dohme F., Nierhaus K. H. Assembly in vitro of the 50 S subunit from Escherichia coli ribosomes: proteins essential for the first heat-dependent conformational change. J Mol Biol. 1977 Sep 25;115(3):513–523. doi: 10.1016/0022-2836(77)90168-1. [DOI] [PubMed] [Google Scholar]
  26. Spillmann S., Nierhaus K. H. The ribosomal protein L24 of Escherichia coli is an assembly protein. J Biol Chem. 1978 Oct 10;253(19):7047–7050. [PubMed] [Google Scholar]
  27. Stelzl U., Spahn C. M., Nierhaus K. H. RNA-protein interactions in ribosomes: in vitro selection from randomly fragmented rRNA. Methods Enzymol. 2000;318:251–268. doi: 10.1016/s0076-6879(00)18057-7. [DOI] [PubMed] [Google Scholar]
  28. Stelzl U., Spahn C. M., Nierhaus K. H. Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: application of a method called SERF. Proc Natl Acad Sci U S A. 2000 Apr 25;97(9):4597–4602. doi: 10.1073/pnas.090009297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Strobel S. A. A chemogenetic approach to RNA function/structure analysis. Curr Opin Struct Biol. 1999 Jun;9(3):346–352. doi: 10.1016/S0959-440X(99)80046-3. [DOI] [PubMed] [Google Scholar]
  30. Triana-Alonso F. J., Dabrowski M., Wadzack J., Nierhaus K. H. Self-coded 3'-extension of run-off transcripts produces aberrant products during in vitro transcription with T7 RNA polymerase. J Biol Chem. 1995 Mar 17;270(11):6298–6307. doi: 10.1074/jbc.270.11.6298. [DOI] [PubMed] [Google Scholar]
  31. Verma S., Eckstein F. Modified oligonucleotides: synthesis and strategy for users. Annu Rev Biochem. 1998;67:99–134. doi: 10.1146/annurev.biochem.67.1.99. [DOI] [PubMed] [Google Scholar]
  32. Vörtler C. S., Fedorova O., Persson T., Kutzke U., Eckstein F. Determination of 2'-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: a nucleotide analogue interference study. RNA. 1998 Nov;4(11):1444–1454. doi: 10.1017/s1355838298980967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zengel J. M., Lindahl L. Domain I of 23S rRNA competes with a paused transcription complex for ribosomal protein L4 of Escherichia coli. Nucleic Acids Res. 1993 May 25;21(10):2429–2435. doi: 10.1093/nar/21.10.2429. [DOI] [PMC free article] [PubMed] [Google Scholar]

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