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. 1998 Nov;4(11):1373–1385. doi: 10.1017/s1355838298981134

Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking.

D Mundus 1, P Wollenzien 1
PMCID: PMC1369710  PMID: 9814758

Abstract

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.

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Selected References

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  1. Atmadja J., Stiege W., Zobawa M., Greuer B., Osswald M., Brimacombe R. The tertiary folding of Escherichia coli 16S RNA, as studied by in situ intra-RNA cross-linking of 30S ribosomal subunits with bis-(2-chloroethyl)-methylamine. Nucleic Acids Res. 1986 Jan 24;14(2):659–673. doi: 10.1093/nar/14.2.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Capel M. S., Engelman D. M., Freeborn B. R., Kjeldgaard M., Langer J. A., Ramakrishnan V., Schindler D. G., Schneider D. K., Schoenborn B. P., Sillers I. Y. A complete mapping of the proteins in the small ribosomal subunit of Escherichia coli. Science. 1987 Dec 4;238(4832):1403–1406. doi: 10.1126/science.3317832. [DOI] [PubMed] [Google Scholar]
  3. Chapman N. M., Noller H. F. Protection of specific sites in 16 S RNA from chemical modification by association of 30 S and 50 S ribosomes. J Mol Biol. 1977 Jan 5;109(1):131–149. doi: 10.1016/s0022-2836(77)80049-1. [DOI] [PubMed] [Google Scholar]
  4. Cimino G. D., Gamper H. B., Isaacs S. T., Hearst J. E. Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry, and biochemistry. Annu Rev Biochem. 1985;54:1151–1193. doi: 10.1146/annurev.bi.54.070185.005443. [DOI] [PubMed] [Google Scholar]
  5. Dragon F., Payant C., Brakier-Gingras L. Mutational and structural analysis of the RNA binding site for Escherichia coli ribosomal protein S7. J Mol Biol. 1994 Nov 18;244(1):74–85. doi: 10.1006/jmbi.1994.1705. [DOI] [PubMed] [Google Scholar]
  6. Dubreuil Y. L., Expert-Bezançon A., Favre A. Conformation and structural fluctuations of a 218 nucleotides long rRNA fragment: 4-thiouridine as an intrinsic photolabelling probe. Nucleic Acids Res. 1991 Jul 11;19(13):3653–3660. doi: 10.1093/nar/19.13.3653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Döring T., Mitchell P., Osswald M., Bochkariov D., Brimacombe R. The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop. EMBO J. 1994 Jun 1;13(11):2677–2685. doi: 10.1002/j.1460-2075.1994.tb06558.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ehresmann C., Moine H., Mougel M., Dondon J., Grunberg-Manago M., Ebel J. P., Ehresmann B. Cross-linking of initiation factor IF3 to Escherichia coli 30S ribosomal subunit by trans-diamminedichloroplatinum(II): characterization of two cross-linking sites in 16S rRNA; a possible way of functioning for IF3. Nucleic Acids Res. 1986 Jun 25;14(12):4803–4821. doi: 10.1093/nar/14.12.4803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ericson G., Minchew P., Wollenzien P. Structural changes in base-paired region 28 in 16 S rRNA close to the decoding region of the 30 S ribosomal subunit are correlated to changes in tRNA binding. J Mol Biol. 1995 Jul 21;250(4):407–419. doi: 10.1006/jmbi.1995.0386. [DOI] [PubMed] [Google Scholar]
  10. Ericson G., Wollenzien P. Use of reverse transcription to determine the exact locations of psoralen photochemical crosslinks in RNA. Anal Biochem. 1988 Oct;174(1):215–223. doi: 10.1016/0003-2697(88)90538-6. [DOI] [PubMed] [Google Scholar]
  11. Fink D. L., Chen R. O., Noller H. F., Altman R. B. Computational methods for defining the allowed conformational space of 16S rRNA based on chemical footprinting data. RNA. 1996 Sep;2(9):851–866. [PMC free article] [PubMed] [Google Scholar]
  12. Frank J. The ribosome at higher resolution--the donut takes shape. Curr Opin Struct Biol. 1997 Apr;7(2):266–272. doi: 10.1016/s0959-440x(97)80035-8. [DOI] [PubMed] [Google Scholar]
  13. Green R., Noller H. F. Ribosomes and translation. Annu Rev Biochem. 1997;66:679–716. doi: 10.1146/annurev.biochem.66.1.679. [DOI] [PubMed] [Google Scholar]
  14. Gutell R. R., Larsen N., Woese C. R. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective. Microbiol Rev. 1994 Mar;58(1):10–26. doi: 10.1128/mr.58.1.10-26.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gutell R. R., Weiser B., Woese C. R., Noller H. F. Comparative anatomy of 16-S-like ribosomal RNA. Prog Nucleic Acid Res Mol Biol. 1985;32:155–216. doi: 10.1016/s0079-6603(08)60348-7. [DOI] [PubMed] [Google Scholar]
  16. Harris M. E., Kazantsev A. V., Chen J. L., Pace N. R. Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking. RNA. 1997 Jun;3(6):561–576. [PMC free article] [PubMed] [Google Scholar]
  17. Herr W., Chapman N. M., Noller H. F. Mechanism of ribosomal subunit association: discrimination of specific sites in 16 S RNA essential for association activity. J Mol Biol. 1979 Jun 5;130(4):433–449. doi: 10.1016/0022-2836(79)90433-9. [DOI] [PubMed] [Google Scholar]
  18. Igloi G. L. Interaction of tRNAs and of phosphorothioate-substituted nucleic acids with an organomercurial. Probing the chemical environment of thiolated residues by affinity electrophoresis. Biochemistry. 1988 May 17;27(10):3842–3849. doi: 10.1021/bi00410a048. [DOI] [PubMed] [Google Scholar]
  19. Isaacs S. T., Shen C. K., Hearst J. E., Rapoport H. Synthesis and characterization of new psoralen derivatives with superior photoreactivity with DNA and RNA. Biochemistry. 1977 Mar 22;16(6):1058–1064. doi: 10.1021/bi00625a005. [DOI] [PubMed] [Google Scholar]
  20. Juzumiene D. I., Shapkina T. G., Wollenzien P. Distribution of cross-links between mRNA analogues and 16 S rRNA in Escherichia coli 70 S ribosomes made under equilibrium conditions and their response to tRNA binding. J Biol Chem. 1995 May 26;270(21):12794–12800. doi: 10.1074/jbc.270.21.12794. [DOI] [PubMed] [Google Scholar]
  21. Kerlavage A. R., Hasan T., Cooperman B. S. Reverse phase high performance liquid chromatography of Escherichia coli ribosomal proteins: standardization of 70 S, 50 S, and 30 S protein chromatograms. J Biol Chem. 1983 May 25;258(10):6313–6318. [PubMed] [Google Scholar]
  22. Krzyzosiak W., Denman R., Nurse K., Hellmann W., Boublik M., Gehrke C. W., Agris P. F., Ofengand J. In vitro synthesis of 16S ribosomal RNA containing single base changes and assembly into a functional 30S ribosome. Biochemistry. 1987 Apr 21;26(8):2353–2364. doi: 10.1021/bi00382a042. [DOI] [PubMed] [Google Scholar]
  23. Lee K., Varma S., SantaLucia J., Jr, Cunningham P. R. In vivo determination of RNA structure-function relationships: analysis of the 790 loop in ribosomal RNA. J Mol Biol. 1997 Jun 27;269(5):732–743. doi: 10.1006/jmbi.1997.1092. [DOI] [PubMed] [Google Scholar]
  24. Malhotra A., Harvey S. C. A quantitative model of the Escherichia coli 16 S RNA in the 30 S ribosomal subunit. J Mol Biol. 1994 Jul 22;240(4):308–340. doi: 10.1006/jmbi.1994.1448. [DOI] [PubMed] [Google Scholar]
  25. McWilliams R. A., Glitz D. G. Localization of a segment of 16S RNA on the surface of the small ribosomal subunit by immune electron microscopy of complementary oligodeoxynucleotides. Biochimie. 1991 Jul-Aug;73(7-8):911–918. doi: 10.1016/0300-9084(91)90132-k. [DOI] [PubMed] [Google Scholar]
  26. Moazed D., Noller H. F. Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA. J Mol Biol. 1990 Jan 5;211(1):135–145. doi: 10.1016/0022-2836(90)90016-F. [DOI] [PubMed] [Google Scholar]
  27. Moazed D., Samaha R. R., Gualerzi C., Noller H. F. Specific protection of 16 S rRNA by translational initiation factors. J Mol Biol. 1995 Apr 28;248(2):207–210. doi: 10.1016/s0022-2836(95)80042-5. [DOI] [PubMed] [Google Scholar]
  28. Moazed D., Stern S., Noller H. F. Rapid chemical probing of conformation in 16 S ribosomal RNA and 30 S ribosomal subunits using primer extension. J Mol Biol. 1986 Feb 5;187(3):399–416. doi: 10.1016/0022-2836(86)90441-9. [DOI] [PubMed] [Google Scholar]
  29. Moazed D., Van Stolk B. J., Douthwaite S., Noller H. F. Interconversion of active and inactive 30 S ribosomal subunits is accompanied by a conformational change in the decoding region of 16 S rRNA. J Mol Biol. 1986 Oct 5;191(3):483–493. doi: 10.1016/0022-2836(86)90143-9. [DOI] [PubMed] [Google Scholar]
  30. Mueller F., Brimacombe R. A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. I. Fitting the RNA to a 3D electron microscopic map at 20 A. J Mol Biol. 1997 Aug 29;271(4):524–544. doi: 10.1006/jmbi.1997.1210. [DOI] [PubMed] [Google Scholar]
  31. Mueller F., Stark H., van Heel M., Rinke-Appel J., Brimacombe R. A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. III. The topography of the functional centre. J Mol Biol. 1997 Aug 29;271(4):566–587. doi: 10.1006/jmbi.1997.1212. [DOI] [PubMed] [Google Scholar]
  32. Muralikrishna P., Wickstrom E. Escherichia coli initiation factor 3 protein binding to 30S ribosomal subunits alters the accessibility of nucleotides within the conserved central region of 16S rRNA. Biochemistry. 1989 Sep 19;28(19):7505–7510. doi: 10.1021/bi00445a002. [DOI] [PubMed] [Google Scholar]
  33. Nierhaus K. H., Dohme F. Total reconstitution of 50 S subunits from Escherichia coli ribosomes. Methods Enzymol. 1979;59:443–449. doi: 10.1016/0076-6879(79)59106-x. [DOI] [PubMed] [Google Scholar]
  34. Noller H. F., Green R., Heilek G., Hoffarth V., Hüttenhofer A., Joseph S., Lee I., Lieberman K., Mankin A., Merryman C. Structure and function of ribosomal RNA. Biochem Cell Biol. 1995 Nov-Dec;73(11-12):997–1009. doi: 10.1139/o95-107. [DOI] [PubMed] [Google Scholar]
  35. Oakes M. I., Lake J. A. DNA-hybridization electron microscopy. Localization of five regions of 16 S rRNA on the surface of 30 S ribosomal subunits. J Mol Biol. 1990 Feb 20;211(4):897–906. doi: 10.1016/0022-2836(90)90082-W. [DOI] [PubMed] [Google Scholar]
  36. Osswald M., Döring T., Brimacombe R. The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site. Nucleic Acids Res. 1995 Nov 25;23(22):4635–4641. doi: 10.1093/nar/23.22.4635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Rinke-Appel J., Jünke N., Stade K., Brimacombe R. The path of mRNA through the Escherichia coli ribosome; site-directed cross-linking of mRNA analogues carrying a photo-reactive label at various points 3' to the decoding site. EMBO J. 1991 Aug;10(8):2195–2202. doi: 10.1002/j.1460-2075.1991.tb07755.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Saffran W. A., Goldenberg M., Cantor C. R. Site-directed psoralen crosslinking of DNA. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4594–4598. doi: 10.1073/pnas.79.15.4594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Santer M., Bennett-Guerrero E., Byahatti S., Czarnecki S., O'Connell D., Meyer M., Khoury J., Cheng X., Schwartz I., McLaughlin J. Base changes at position 792 of Escherichia coli 16S rRNA affect assembly of 70S ribosomes. Proc Natl Acad Sci U S A. 1990 May;87(10):3700–3704. doi: 10.1073/pnas.87.10.3700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Serdyuk I. N., Agalarov S. C., Sedelnikova S. E., Spirin A. S., May R. P. Shape and compactness of the isolated ribosomal 16 S RNA and its complexes with ribosomal proteins. J Mol Biol. 1983 Sep 15;169(2):409–425. doi: 10.1016/s0022-2836(83)80058-8. [DOI] [PubMed] [Google Scholar]
  42. Sergiev P. V., Lavrik I. N., Wlasoff V. A., Dokudovskaya S. S., Dontsova O. A., Bogdanov A. A., Brimacombe R. The path of mRNA through the bacterial ribosome: a site-directed crosslinking study using new photoreactive derivatives of guanosine and uridine. RNA. 1997 May;3(5):464–475. [PMC free article] [PubMed] [Google Scholar]
  43. Stade K., Rinke-Appel J., Brimacombe R. Site-directed cross-linking of mRNA analogues to the Escherichia coli ribosome; identification of 30S ribosomal components that can be cross-linked to the mRNA at various points 5' with respect to the decoding site. Nucleic Acids Res. 1989 Dec 11;17(23):9889–9908. doi: 10.1093/nar/17.23.9889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stark H., Orlova E. V., Rinke-Appel J., Jünke N., Mueller F., Rodnina M., Wintermeyer W., Brimacombe R., van Heel M. Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy. Cell. 1997 Jan 10;88(1):19–28. doi: 10.1016/s0092-8674(00)81854-1. [DOI] [PubMed] [Google Scholar]
  45. Stern S., Powers T., Changchien L. M., Noller H. F. RNA-protein interactions in 30S ribosomal subunits: folding and function of 16S rRNA. Science. 1989 May 19;244(4906):783–790. doi: 10.1126/science.2658053. [DOI] [PubMed] [Google Scholar]
  46. Stern S., Weiser B., Noller H. F. Model for the three-dimensional folding of 16 S ribosomal RNA. J Mol Biol. 1988 Nov 20;204(2):447–481. doi: 10.1016/0022-2836(88)90588-8. [DOI] [PubMed] [Google Scholar]
  47. Tapprich W. E., Goss D. J., Dahlberg A. E. Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4927–4931. doi: 10.1073/pnas.86.13.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tapprich W. E., Hill W. E. Involvement of bases 787-795 of Escherichia coli 16S ribosomal RNA in ribosomal subunit association. Proc Natl Acad Sci U S A. 1986 Feb;83(3):556–560. doi: 10.1073/pnas.83.3.556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Teare J., Wollenzien P. Specificity of site directed psoralen addition to RNA. Nucleic Acids Res. 1989 May 11;17(9):3359–3372. doi: 10.1093/nar/17.9.3359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Teare J., Wollenzien P. The structure of a pre-mRNA molecule in solution determined with a site directed cross-linking reagent. Nucleic Acids Res. 1990 Feb 25;18(4):855–864. doi: 10.1093/nar/18.4.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wilms C., Noah J. W., Zhong D., Wollenzien P. Exact determination of UV-induced crosslinks in 16S ribosomal RNA in 30S ribosomal subunits. RNA. 1997 Jun;3(6):602–612. [PMC free article] [PubMed] [Google Scholar]
  52. Wilms C., Wollenzien P. Purification of RNA from polyacrylamide gels by ultracentrifugation. Anal Biochem. 1994 Aug 15;221(1):204–205. doi: 10.1006/abio.1994.1399. [DOI] [PubMed] [Google Scholar]
  53. Woese C. R., Magrum L. J., Gupta R., Siegel R. B., Stahl D. A., Kop J., Crawford N., Brosius J., Gutell R., Hogan J. J. Secondary structure model for bacterial 16S ribosomal RNA: phylogenetic, enzymatic and chemical evidence. Nucleic Acids Res. 1980 May 24;8(10):2275–2293. doi: 10.1093/nar/8.10.2275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wollenzien P. L. Isolation and identification of RNA cross-links. Methods Enzymol. 1988;164:319–329. doi: 10.1016/s0076-6879(88)64052-3. [DOI] [PubMed] [Google Scholar]
  55. Zamir A., Miskin R., Vogel Z., Elson D. The inactivation and reactivation of Escherichia coli ribosomes. Methods Enzymol. 1974;30:406–426. doi: 10.1016/0076-6879(74)30042-0. [DOI] [PubMed] [Google Scholar]
  56. von Ahsen U., Noller H. F. Identification of bases in 16S rRNA essential for tRNA binding at the 30S ribosomal P site. Science. 1995 Jan 13;267(5195):234–237. doi: 10.1126/science.7528943. [DOI] [PubMed] [Google Scholar]

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