Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Jun 15;24(12):2352–2359. doi: 10.1093/nar/24.12.2352

Complementation of RNA binding site mutations in MS2 coat protein heterodimers.

D S Peabody 1, F Lim 1
PMCID: PMC145953  PMID: 8710507

Abstract

The coat protein of bacteriophage MS2 functions as a symmetric dimer to bind an asymmetric RNA hairpin. This implies the existence of two equivalent RNA binding sites related to one another by a 2-fold symmetry axis. In this view the symmetric binding site defined by mutations conferring the repressor-defective phenotype is a composite picture of these two asymmetric sites. In order to determine whether the RNA ligand interacts with amino acid residues on both subunits of the dimer and in the hope of constructing a functional map of the RNA binding site, we performed heterodimer complementation experiments. Taking advantage of the physical proximity of their N- and C-termini, the two subunits of the dimer were genetically fused, producing a duplicated coat protein which folds normally and allows the construction of the functional equivalent of obligatory heterodimers containing all possible pairwise combinations of the repressor-defective mutations. The restoration of repressor function in certain heterodimers shows that a single RNA molecule interacts with both subunits of the dimer and allows the construction of a functional map of the binding site.

Full Text

The Full Text of this article is available as a PDF (121.4 KB).

Selected References

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

  1. Beckett D., Uhlenbeck O. C. Ribonucleoprotein complexes of R17 coat protein and a translational operator analog. J Mol Biol. 1988 Dec 20;204(4):927–938. doi: 10.1016/0022-2836(88)90052-6. [DOI] [PubMed] [Google Scholar]
  2. Bernardi A., Spahr P. F. Nucleotide sequence at the binding site for coat protein on RNA of bacteriophage R17. Proc Natl Acad Sci U S A. 1972 Oct;69(10):3033–3037. doi: 10.1073/pnas.69.10.3033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  4. Carey J., Lowary P. T., Uhlenbeck O. C. Interaction of R17 coat protein with synthetic variants of its ribonucleic acid binding site. Biochemistry. 1983 Sep 27;22(20):4723–4730. doi: 10.1021/bi00289a017. [DOI] [PubMed] [Google Scholar]
  5. Draper D. E., White S. A., Kean J. M. Preparation of specific ribosomal RNA fragments. Methods Enzymol. 1988;164:221–237. doi: 10.1016/s0076-6879(88)64045-6. [DOI] [PubMed] [Google Scholar]
  6. Golmohammadi R., Valegård K., Fridborg K., Liljas L. The refined structure of bacteriophage MS2 at 2.8 A resolution. J Mol Biol. 1993 Dec 5;234(3):620–639. doi: 10.1006/jmbi.1993.1616. [DOI] [PubMed] [Google Scholar]
  7. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  8. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  9. Lim F., Peabody D. S. Mutations that increase the affinity of a translational repressor for RNA. Nucleic Acids Res. 1994 Sep 11;22(18):3748–3752. doi: 10.1093/nar/22.18.3748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lim F., Spingola M., Peabody D. S. Altering the RNA binding specificity of a translational repressor. J Biol Chem. 1994 Mar 25;269(12):9006–9010. [PubMed] [Google Scholar]
  11. Ni C. Z., Syed R., Kodandapani R., Wickersham J., Peabody D. S., Ely K. R. Crystal structure of the MS2 coat protein dimer: implications for RNA binding and virus assembly. Structure. 1995 Mar 15;3(3):255–263. doi: 10.1016/S0969-2126(01)00156-3. [DOI] [PubMed] [Google Scholar]
  12. Peabody D. S., Ely K. R. Control of translational repression by protein-protein interactions. Nucleic Acids Res. 1992 Apr 11;20(7):1649–1655. doi: 10.1093/nar/20.7.1649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Peabody D. S. The RNA binding site of bacteriophage MS2 coat protein. EMBO J. 1993 Feb;12(2):595–600. doi: 10.1002/j.1460-2075.1993.tb05691.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Peabody D. S. Translational repression by bacteriophage MS2 coat protein expressed from a plasmid. A system for genetic analysis of a protein-RNA interaction. J Biol Chem. 1990 Apr 5;265(10):5684–5689. [PubMed] [Google Scholar]
  15. Reddy P., Peterkofsky A., McKenney K. Translational efficiency of the Escherichia coli adenylate cyclase gene: mutating the UUG initiation codon to GUG or AUG results in increased gene expression. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5656–5660. doi: 10.1073/pnas.82.17.5656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Valegård K., Liljas L., Fridborg K., Unge T. The three-dimensional structure of the bacterial virus MS2. Nature. 1990 May 3;345(6270):36–41. doi: 10.1038/345036a0. [DOI] [PubMed] [Google Scholar]
  17. Valegård K., Murray J. B., Stockley P. G., Stonehouse N. J., Liljas L. Crystal structure of an RNA bacteriophage coat protein-operator complex. Nature. 1994 Oct 13;371(6498):623–626. doi: 10.1038/371623a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES