Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Jul 15;25(14):2835–2840. doi: 10.1093/nar/25.14.2835

Determinants of a protein-induced RNA switch in the large domain of signal recognition particle identified by systematic-site directed mutagenesis.

K Gowda 1, C Zwieb 1
PMCID: PMC146806  PMID: 9207032

Abstract

Signal recognition particle (SRP) is a ribonucleoprotein complex that associates with ribosomes to promote the co-translational translocation of proteins across biological membranes. Human SRP RNA molecules exist in two distinct conformations, SR-A and SR-B, which may exchange during the assembly of the particle or could play a functional role in the SRP cycle. We have used systematic site-directed mutagenesis of the SRP RNA to determine the electrophoretic mobilities of altered RNA molecules, and we have identified the nucleotides that avert the formation of the two conformers. The conformer behavior of the various RNAs was addressed quantitatively by calculating a value zeta as an indicator of conformational variability. Single loose A-like forms were induced by changes in helix 5 [nucleotides (nt) at positions 111-128 or 222-231], helix 6 (nt at positions 141-150) and helix 7 (nt at position 169 and 170), whereas other mutations in helix 6 and helix 8 preserved the conformational variability of the mutant RNA molecules. The more compact B-like form was induced only when a small region (129-CAAUAU-134), located in the 5'-proximal portion of helix 6, was altered. Since this region is part of the binding site for SRP19, we suggest that protein SRP19 uses nucleotides at 129-134 to trigger the formation of the favored SR-B-form, and thus directs an early step in the hierarchical assembly of the large SRP domain.

Full Text

The Full Text of this article is available as a PDF (134.2 KB).

Selected References

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

  1. Andreazzoli M., Gerbi S. A. Changes in 7SL RNA conformation during the signal recognition particle cycle. EMBO J. 1991 Apr;10(4):767–777. doi: 10.1002/j.1460-2075.1991.tb08008.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andrews D. W., Walter P., Ottensmeyer F. P. Evidence for an extended 7SL RNA structure in the signal recognition particle. EMBO J. 1987 Nov;6(11):3471–3477. doi: 10.1002/j.1460-2075.1987.tb02671.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  4. Butler E. T., Chamberlin M. J. Bacteriophage SP6-specific RNA polymerase. I. Isolation and characterization of the enzyme. J Biol Chem. 1982 May 25;257(10):5772–5778. [PubMed] [Google Scholar]
  5. Cech T. R., Zaug A. J., Grabowski P. J. In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence. Cell. 1981 Dec;27(3 Pt 2):487–496. doi: 10.1016/0092-8674(81)90390-1. [DOI] [PubMed] [Google Scholar]
  6. Gast F. U., Hagerman P. J. Electrophoretic and hydrodynamic properties of duplex ribonucleic acid molecules transcribed in vitro: evidence that A-tracts do not generate curvature in RNA. Biochemistry. 1991 Apr 30;30(17):4268–4277. doi: 10.1021/bi00231a024. [DOI] [PubMed] [Google Scholar]
  7. Gowda K., Chittenden K., Zwieb C. Binding site of the M-domain of human protein SRP54 determined by systematic site-directed mutagenesis of signal recognition particle RNA. Nucleic Acids Res. 1997 Jan 15;25(2):388–394. doi: 10.1093/nar/25.2.388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Guerrier-Takada C., Gardiner K., Marsh T., Pace N., Altman S. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell. 1983 Dec;35(3 Pt 2):849–857. doi: 10.1016/0092-8674(83)90117-4. [DOI] [PubMed] [Google Scholar]
  9. Gundelfinger E. D., Di Carlo M., Zopf D., Melli M. Structure and evolution of the 7SL RNA component of the signal recognition particle. EMBO J. 1984 Oct;3(10):2325–2332. doi: 10.1002/j.1460-2075.1984.tb02134.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gundelfinger E. D., Krause E., Melli M., Dobberstein B. The organization of the 7SL RNA in the signal recognition particle. Nucleic Acids Res. 1983 Nov 11;11(21):7363–7374. doi: 10.1093/nar/11.21.7363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Larsen N., Zwieb C. SRP-RNA sequence alignment and secondary structure. Nucleic Acids Res. 1991 Jan 25;19(2):209–215. doi: 10.1093/nar/19.2.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Larsen N., Zwieb C. The Signal Recognition Particle Database (SRPDB). Nucleic Acids Res. 1996 Jan 1;24(1):80–81. doi: 10.1093/nar/24.1.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lingelbach K., Zwieb C., Webb J. R., Marshallsay C., Hoben P. J., Walter P., Dobberstein B. Isolation and characterization of a cDNA clone encoding the 19 kDa protein of signal recognition particle (SRP): expression and binding to 7SL RNA. Nucleic Acids Res. 1988 Oct 25;16(20):9431–9442. doi: 10.1093/nar/16.20.9431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lütcke H., Prehn S., Ashford A. J., Remus M., Frank R., Dobberstein B. Assembly of the 68- and 72-kD proteins of signal recognition particle with 7S RNA. J Cell Biol. 1993 Jun;121(5):977–985. doi: 10.1083/jcb.121.5.977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lütcke H. Signal recognition particle (SRP), a ubiquitous initiator of protein translocation. Eur J Biochem. 1995 Mar 15;228(3):531–550. doi: 10.1111/j.1432-1033.1995.tb20293.x. [DOI] [PubMed] [Google Scholar]
  16. Massire C., Gaspin C., Westhof E. DRAWNA: a program for drawing schematic views of nucleic acids. J Mol Graph. 1994 Sep;12(3):201-6, 196. doi: 10.1016/0263-7855(94)80088-x. [DOI] [PubMed] [Google Scholar]
  17. Nelson R. M., Long G. L. A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal Biochem. 1989 Jul;180(1):147–151. doi: 10.1016/0003-2697(89)90103-6. [DOI] [PubMed] [Google Scholar]
  18. Noller H. F., Hoffarth V., Zimniak L. Unusual resistance of peptidyl transferase to protein extraction procedures. Science. 1992 Jun 5;256(5062):1416–1419. doi: 10.1126/science.1604315. [DOI] [PubMed] [Google Scholar]
  19. Siegel V., Walter P. Binding sites of the 19-kDa and 68/72-kDa signal recognition particle (SRP) proteins on SRP RNA as determined in protein-RNA "footprinting". Proc Natl Acad Sci U S A. 1988 Mar;85(6):1801–1805. doi: 10.1073/pnas.85.6.1801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tang R. S., Draper D. E. Bend and helical twist associated with a symmetric internal loop from 5S ribosomal RNA. Biochemistry. 1994 Aug 23;33(33):10089–10093. doi: 10.1021/bi00199a036. [DOI] [PubMed] [Google Scholar]
  21. Walker K. P., 3rd, Black S. D., Zwieb C. Cooperative assembly of signal recognition particle RNA with protein SRP19. Biochemistry. 1995 Sep 19;34(37):11989–11997. doi: 10.1021/bi00037a041. [DOI] [PubMed] [Google Scholar]
  22. Walter P., Blobel G. Disassembly and reconstitution of signal recognition particle. Cell. 1983 Sep;34(2):525–533. doi: 10.1016/0092-8674(83)90385-9. [DOI] [PubMed] [Google Scholar]
  23. Wolffe A. P. Transcription fraction TFIIIC can regulate differential Xenopus 5S RNA gene transcription in vitro. EMBO J. 1988 Apr;7(4):1071–1079. doi: 10.1002/j.1460-2075.1988.tb02915.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wu H. M., Crothers D. M. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. doi: 10.1038/308509a0. [DOI] [PubMed] [Google Scholar]
  25. Zwieb C. Interaction of protein SRP19 with signal recognition particle RNA lacking individual RNA-helices. Nucleic Acids Res. 1991 Jun 11;19(11):2955–2960. doi: 10.1093/nar/19.11.2955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zwieb C., Kim J., Adhya S. DNA bending by negative regulatory proteins: Gal and Lac repressors. Genes Dev. 1989 May;3(5):606–611. doi: 10.1101/gad.3.5.606. [DOI] [PubMed] [Google Scholar]
  27. Zwieb C., Müller F., Larsen N. Comparative analysis of tertiary structure elements in signal recognition particle RNA. Fold Des. 1996;1(4):315–324. doi: 10.1016/S1359-0278(96)00044-2. [DOI] [PubMed] [Google Scholar]
  28. Zwieb C. Recognition of a tetranucleotide loop of signal recognition particle RNA by protein SRP19. J Biol Chem. 1992 Aug 5;267(22):15650–15656. [PubMed] [Google Scholar]
  29. Zwieb C. The secondary structure of the 7SL RNA in the signal recognition particle: functional implications. Nucleic Acids Res. 1985 Sep 11;13(17):6105–6124. doi: 10.1093/nar/13.17.6105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zwieb C., Ullu E. Identification of dynamic sequences in the central domain of 7SL RNA. Nucleic Acids Res. 1986 Jun 11;14(11):4639–4657. doi: 10.1093/nar/14.11.4639. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES