Skip to main content
NCBI home page
RNA logoLink to RNA
. 1998 Nov;4(11):1455–1466. doi: 10.1017/s1355838298981079

Analysis of the conformation of the 3' major domain of Escherichia coli16S ribosomal RNA using site-directed photoaffinity crosslinking.

A Montpetit 1, C Payant 1, J M Nolan 1, L Brakier-Gingras 1
PMCID: PMC1369717  PMID: 9814765

Abstract

The 3' major domain of Escherichia coli 16S rRNA, which occupies the head of the small ribosomal subunit, is involved in several functions of the ribosome. We have used a site-specific crosslinking procedure to gain further insights into the higher-order structure of this domain. Circularly permuted RNAs were used to introduce an azidophenacyl group at specific positions within the 3' major domain. Crosslinks were generated in a high-ionic strength buffer that has been used for ribosome reconstitution studies and so enables the RNA to adopt a structure recognized by ribosomal proteins. The crosslinking sites were identified by primer extension and confirmed by assessing the mobility of the crosslinked RNA lariats in denaturing polyacrylamide gels. Eight crosslinks were characterized. Among them, one crosslink demonstrates that helix 28 is proximal to the top of helix 34, and two others show that the 1337 region, located in an internal loop at the junction of helices 29, 30, 41, and 42, is proximal to the center of helix 30 and to a segment connecting helix 28 to helix 29. These relationships of vicinity have previously been observed in native 30S subunits, which suggests that the free domain adopts a conformation similar to that within the 30S subunit. Furthermore, crosslinks were obtained in helix 34, which suggest that the upper and lower portions of this helix are in close proximity.

Full Text

The Full Text of this article is available as a PDF (913.6 KB).

Selected References

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

  1. Agalarov S. C., Zheleznyakova E. N., Selivanova O. M., Zheleznaya L. A., Matvienko N. I., Vasiliev V. D., Spirin A. S. In vitro assembly of a ribonucleoprotein particle corresponding to the platform domain of the 30S ribosomal subunit. Proc Natl Acad Sci U S A. 1998 Feb 3;95(3):999–1003. doi: 10.1073/pnas.95.3.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander R. W., Cooperman B. S. Ribosomal proteins neighboring 23 S rRNA nucleotides 803-811 within the 50 S subunit. Biochemistry. 1998 Feb 10;37(6):1714–1721. doi: 10.1021/bi972280+. [DOI] [PubMed] [Google Scholar]
  3. Arkov A. L., Freistroffer D. V., Ehrenberg M., Murgola E. J. Mutations in RNAs of both ribosomal subunits cause defects in translation termination. EMBO J. 1998 Mar 2;17(5):1507–1514. doi: 10.1093/emboj/17.5.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ban N., Freeborn B., Nissen P., Penczek P., Grassucci R. A., Sweet R., Frank J., Moore P. B., Steitz T. A. A 9 A resolution X-ray crystallographic map of the large ribosomal subunit. Cell. 1998 Jun 26;93(7):1105–1115. doi: 10.1016/s0092-8674(00)81455-5. [DOI] [PubMed] [Google Scholar]
  5. Baranov P. V., Gurvich O. L., Bogdanov A. A., Brimacombe R., Dontsova O. A. New features of 23S ribosomal RNA folding: the long helix 41-42 makes a "U-turn" inside the ribosome. RNA. 1998 Jun;4(6):658–668. doi: 10.1017/s1355838298980104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baranov P. V., Sergiev P. V., Dontsova O. A., Bogdanov A. A., Brimacombe R. The Database of Ribosomal Cross links (DRC). Nucleic Acids Res. 1998 Jan 1;26(1):187–189. doi: 10.1093/nar/26.1.187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Baudin F., Mougel M., Romby P., Eyermann F., Ebel J. P., Ehresmann B., Ehresmann C. Probing the phosphates of the Escherichia coli ribosomal 16S RNA in its naked form, in the 30S subunit, and in the 70S ribosome. Biochemistry. 1989 Jul 11;28(14):5847–5855. doi: 10.1021/bi00440a022. [DOI] [PubMed] [Google Scholar]
  8. Bilgin N., Richter A. A., Ehrenberg M., Dahlberg A. E., Kurland C. G. Ribosomal RNA and protein mutants resistant to spectinomycin. EMBO J. 1990 Mar;9(3):735–739. doi: 10.1002/j.1460-2075.1990.tb08167.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brimacombe R. RNA-protein interactions in the Escherichia coli ribosome. Biochimie. 1991 Jul-Aug;73(7-8):927–936. doi: 10.1016/0300-9084(91)90134-m. [DOI] [PubMed] [Google Scholar]
  10. Brink M. F., Brink G., Verbeet M. P., de Boer H. A. Spectinomycin interacts specifically with the residues G1064 and C1192 in 16S rRNA, thereby potentially freezing this molecule into an inactive conformation. Nucleic Acids Res. 1994 Feb 11;22(3):325–331. doi: 10.1093/nar/22.3.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bucklin D. J., van Waes M. A., Bullard J. M., Hill W. E. Cleavage of 16S rRNA within the ribosome by mRNA modified in the A-site codon with phenanthroline-Cu(II). Biochemistry. 1997 Jul 1;36(26):7951–7957. doi: 10.1021/bi9624954. [DOI] [PubMed] [Google Scholar]
  12. Burgin A. B., Pace N. R. Mapping the active site of ribonuclease P RNA using a substrate containing a photoaffinity agent. EMBO J. 1990 Dec;9(12):4111–4118. doi: 10.1002/j.1460-2075.1990.tb07633.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cate J. H., Gooding A. R., Podell E., Zhou K., Golden B. L., Kundrot C. E., Cech T. R., Doudna J. A. Crystal structure of a group I ribozyme domain: principles of RNA packing. Science. 1996 Sep 20;273(5282):1678–1685. doi: 10.1126/science.273.5282.1678. [DOI] [PubMed] [Google Scholar]
  14. Chen J. L., Nolan J. M., Harris M. E., Pace N. R. Comparative photocross-linking analysis of the tertiary structures of Escherichia coli and Bacillus subtilis RNase P RNAs. EMBO J. 1998 Mar 2;17(5):1515–1525. doi: 10.1093/emboj/17.5.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Correll C. C., Freeborn B., Moore P. B., Steitz T. A. Metals, motifs, and recognition in the crystal structure of a 5S rRNA domain. Cell. 1997 Nov 28;91(5):705–712. doi: 10.1016/s0092-8674(00)80457-2. [DOI] [PubMed] [Google Scholar]
  16. Dallas A., Moore P. B. The loop E-loop D region of Escherichia coli 5S rRNA: the solution structure reveals an unusual loop that may be important for binding ribosomal proteins. Structure. 1997 Dec 15;5(12):1639–1653. doi: 10.1016/s0969-2126(97)00311-0. [DOI] [PubMed] [Google Scholar]
  17. Doudna J. A., Cate J. H. RNA structure: crystal clear? Curr Opin Struct Biol. 1997 Jun;7(3):310–316. doi: 10.1016/s0959-440x(97)80045-0. [DOI] [PubMed] [Google Scholar]
  18. Dragon F., Brakier-Gingras L. Interaction of Escherichia coli ribosomal protein S7 with 16S rRNA. Nucleic Acids Res. 1993 Mar 11;21(5):1199–1203. doi: 10.1093/nar/21.5.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Fourmy D., Recht M. I., Blanchard S. C., Puglisi J. D. Structure of the A site of Escherichia coli 16S ribosomal RNA complexed with an aminoglycoside antibiotic. Science. 1996 Nov 22;274(5291):1367–1371. doi: 10.1126/science.274.5291.1367. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Joseph S., Weiser B., Noller H. F. Mapping the inside of the ribosome with an RNA helical ruler. Science. 1997 Nov 7;278(5340):1093–1098. doi: 10.1126/science.278.5340.1093. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Laughrea M., Tam J. In vivo chemical footprinting of the Escherichia coli ribosome. Biochemistry. 1992 Dec 8;31(48):12035–12041. doi: 10.1021/bi00163a011. [DOI] [PubMed] [Google Scholar]
  28. Makosky P. C., Dahlberg A. E. Spectinomycin resistance at site 1192 in 16S ribosomal RNA of E. coli: an analysis of three mutants. Biochimie. 1987 Aug;69(8):885–889. doi: 10.1016/0300-9084(87)90216-1. [DOI] [PubMed] [Google Scholar]
  29. Melançon P., Gravel M., Boileau G., Brakier-Gingras L. Reassembly of active 30S ribosomal subunits with an unmethylated in vitro transcribed 16S rRNA. Biochem Cell Biol. 1987 Dec;65(12):1022–1030. doi: 10.1139/o87-134. [DOI] [PubMed] [Google Scholar]
  30. Milligan J. F., Uhlenbeck O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 1989;180:51–62. doi: 10.1016/0076-6879(89)80091-6. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Moine H., Dahlberg A. E. Mutations in helix 34 of Escherichia coli 16 S ribosomal RNA have multiple effects on ribosome function and synthesis. J Mol Biol. 1994 Oct 28;243(3):402–412. doi: 10.1006/jmbi.1994.1668. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Nitta I., Kamada Y., Noda H., Ueda T., Watanabe K. Reconstitution of peptide bond formation with Escherichia coli 23S ribosomal RNA domains. Science. 1998 Jul 31;281(5377):666–669. doi: 10.1126/science.281.5377.666. [DOI] [PubMed] [Google Scholar]
  36. Nitta I., Ueda T., Watanabe K. Possible involvement of Escherichia coli 23S ribosomal RNA in peptide bond formation. RNA. 1998 Mar;4(3):257–267. [PMC free article] [PubMed] [Google Scholar] [Retracted]
  37. Nolan J. M., Burke D. H., Pace N. R. Circularly permuted tRNAs as specific photoaffinity probes of ribonuclease P RNA structure. Science. 1993 Aug 6;261(5122):762–765. doi: 10.1126/science.7688143. [DOI] [PubMed] [Google Scholar]
  38. O'Connor M., Thomas C. L., Zimmermann R. A., Dahlberg A. E. Decoding fidelity at the ribosomal A and P sites: influence of mutations in three different regions of the decoding domain in 16S rRNA. Nucleic Acids Res. 1997 Mar 15;25(6):1185–1193. doi: 10.1093/nar/25.6.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Oehler R., Polacek N., Steiner G., Barta A. Interaction of tetracycline with RNA: photoincorporation into ribosomal RNA of Escherichia coli. Nucleic Acids Res. 1997 Mar 15;25(6):1219–1224. doi: 10.1093/nar/25.6.1219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Orr J. W., Hagerman P. J., Williamson J. R. Protein and Mg(2+)-induced conformational changes in the S15 binding site of 16 S ribosomal RNA. J Mol Biol. 1998 Jan 23;275(3):453–464. doi: 10.1006/jmbi.1997.1489. [DOI] [PubMed] [Google Scholar]
  41. Puglisi E. V., Green R., Noller H. F., Puglisi J. D. Structure of a conserved RNA component of the peptidyl transferase centre. Nat Struct Biol. 1997 Oct;4(10):775–778. doi: 10.1038/nsb1097-775. [DOI] [PubMed] [Google Scholar]
  42. Purohit P., Stern S. Interactions of a small RNA with antibiotic and RNA ligands of the 30S subunit. Nature. 1994 Aug 25;370(6491):659–662. doi: 10.1038/370659a0. [DOI] [PubMed] [Google Scholar]
  43. Ramos A., Gubser C. C., Varani G. Recent solution structures of RNA and its complexes with drugs, peptides and proteins. Curr Opin Struct Biol. 1997 Jun;7(3):317–323. doi: 10.1016/s0959-440x(97)80046-2. [DOI] [PubMed] [Google Scholar]
  44. Rinke-Appel J., Jünke N., Brimacombe R., Dukudovskaya S., Dontsova O., Bogdanov A. Site-directed cross-linking of mRNA analogues to 16S ribosomal RNA; a complete scan of cross-links from all positions between '+1' and '+16' on the mRNA, downstream from the decoding site. Nucleic Acids Res. 1993 Jun 25;21(12):2853–2859. doi: 10.1093/nar/21.12.2853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Samaha R. R., O'Brien B., O'Brien T. W., Noller H. F. Independent in vitro assembly of a ribonucleoprotein particle containing the 3' domain of 16S rRNA. Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):7884–7888. doi: 10.1073/pnas.91.17.7884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. Stiege W., Kosack M., Stade K., Brimacombe R. Intra-RNA cross-linking in Escherichia coli 30S ribosomal subunits: selective isolation of cross-linked products by hybridization to specific cDNA fragments. Nucleic Acids Res. 1988 May 25;16(10):4315–4329. doi: 10.1093/nar/16.10.4315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Szewczak A. A., Moore P. B. The sarcin/ricin loop, a modular RNA. J Mol Biol. 1995 Mar 17;247(1):81–98. doi: 10.1006/jmbi.1994.0124. [DOI] [PubMed] [Google Scholar]
  51. Thygesen J., Weinstein S., Franceschi F., Yonath A. The suitability of multi-metal clusters for phasing in crystallography of large macromolecular assemblies. Structure. 1996 May 15;4(5):513–518. doi: 10.1016/s0969-2126(96)00057-3. [DOI] [PubMed] [Google Scholar]
  52. Wakao H., Romby P., Ebel J. P., Grunberg-Manago M., Ehresmann C., Ehresmann B. Topography of the Escherichia coli ribosomal 30S subunit-initiation factor 2 complex. Biochimie. 1991 Jul-Aug;73(7-8):991–1000. doi: 10.1016/0300-9084(91)90140-v. [DOI] [PubMed] [Google Scholar]
  53. Weitzmann C. J., Cunningham P. R., Nurse K., Ofengand J. Chemical evidence for domain assembly of the Escherichia coli 30S ribosome. FASEB J. 1993 Jan;7(1):177–180. doi: 10.1096/fasebj.7.1.7916699. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Wilson K. S., Noller H. F. Mapping the position of translational elongation factor EF-G in the ribosome by directed hydroxyl radical probing. Cell. 1998 Jan 9;92(1):131–139. doi: 10.1016/s0092-8674(00)80905-8. [DOI] [PubMed] [Google Scholar]
  56. Xing Y., Draper D. E. Stabilization of a ribosomal RNA tertiary structure by ribosomal protein L11. J Mol Biol. 1995 Jun 2;249(2):319–331. doi: 10.1006/jmbi.1995.0299. [DOI] [PubMed] [Google Scholar]
  57. Zawadzki V., Gross H. J. Rapid and simple purification of T7 RNA polymerase. Nucleic Acids Res. 1991 Apr 25;19(8):1948–1948. doi: 10.1093/nar/19.8.1948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. 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]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES