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. 1997 Apr 15;25(8):1649–1657. doi: 10.1093/nar/25.8.1649

Direct genetic selection of two classes of R17/MS2 coat proteins with altered capsid assembly properties and expanded RNA-binding activities.

S Wang 1, H L True 1, E M Seitz 1, K A Bennett 1, D E Fouts 1, J F Gardner 1, D W Celander 1
PMCID: PMC146620  PMID: 9092675

Abstract

RNA challenge phages are derivatives of bacteriophage P22 that enable direct genetic selection for a specific RNA-protein interaction. The bacteriophage P22 R17 encodes a wild-type R17 operator site and undergoes lysogenic development following infection of susceptible bacterial strains that express the R17/MS2 coat protein. A P22 R17 derivative with an OcRNA site (P22 R17 [A(-10)U]) develops lytically following infection of these strains. RNA challenge phages can be used to isolate second-site coat protein suppressors that recognize an OcRNA sequence by selecting for lysogens with a P22 R17 [Oc] phage derivative. The bacteriophage derivative P22 R17 [A(-10)U] was used in one such scheme to isolate two classes of genes that encode R17 coat proteins with altered capsid assembly properties and expanded RNA-binding characteristics. These mutations map outside the RNA-binding surface and include amino acid substitutions that interfere with interactions between coat protein dimers in the formation of the stable phage capsid. One class of mutants encodes substitutions at the highly conserved first and second positions of the mature coat protein. N-terminal sequence analysis of these mutants reveals that coat proteins with substitutions only at position 1 are defective in post-translational processing of the initiator methionine. All selected proteins possess expanded RNA-binding properties since they direct efficient lysogen formation for P22 R17 and P22 R17 [A(-10)U]; however, bacterial strains that express the protein mutants remain sensitive to lytic infection by other P22 R17 [Oc] bacteriophages. The described selection strategy provides a novel genetic approach to dissecting protein structure within RNA-binding proteins.

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Selected References

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

  1. Ben-Bassat A., Bauer K., Chang S. Y., Myambo K., Boosman A., Chang S. Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure. J Bacteriol. 1987 Feb;169(2):751–757. doi: 10.1128/jb.169.2.751-757.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berkhout B., Schmidt B. F., van Strien A., van Boom J., van Westrenen J., van Duin J. Lysis gene of bacteriophage MS2 is activated by translation termination at the overlapping coat gene. J Mol Biol. 1987 Jun 5;195(3):517–524. doi: 10.1016/0022-2836(87)90180-x. [DOI] [PubMed] [Google Scholar]
  3. Berkhout B., van Duin J. Mechanism of translational coupling between coat protein and replicase genes of RNA bacteriophage MS2. Nucleic Acids Res. 1985 Oct 11;13(19):6955–6967. doi: 10.1093/nar/13.19.6955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Cadwell R. C., Joyce G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 1992 Aug;2(1):28–33. doi: 10.1101/gr.2.1.28. [DOI] [PubMed] [Google Scholar]
  6. Carey J., Cameron V., de Haseth P. L., Uhlenbeck O. C. Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site. Biochemistry. 1983 May 24;22(11):2601–2610. doi: 10.1021/bi00280a002. [DOI] [PubMed] [Google Scholar]
  7. Eggen K., Nathans D. Regulation of protein synthesis directed by coliphage MS2 RNA. II. In vitro repression by phage coat protein. J Mol Biol. 1969 Jan;39(2):293–305. doi: 10.1016/0022-2836(69)90318-0. [DOI] [PubMed] [Google Scholar]
  8. Eggen K., Oeschger M. P., Nathans D. Cell-free protein synthesis directed by colphage MS2 RNA: sequential synthesis of specific phage proteins. Biochem Biophys Res Commun. 1967 Aug 23;28(4):587–597. doi: 10.1016/0006-291x(67)90354-3. [DOI] [PubMed] [Google Scholar]
  9. GESTELAND R. F., BOEDTKER H. SOME PHYSICAL PROPERTIES OF BACTERIOPHAGE R17 AND ITS RIBONUCLEIC ACID. J Mol Biol. 1964 Apr;8:496–507. doi: 10.1016/s0022-2836(64)80007-3. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Hindley J., Staples D. H. Sequence of a ribosome binding site in bacteriophage Q-beta-RNA. Nature. 1969 Dec 6;224(5223):964–967. doi: 10.1038/224964a0. [DOI] [PubMed] [Google Scholar]
  12. Hirel P. H., Schmitter M. J., Dessen P., Fayat G., Blanquet S. Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8247–8251. doi: 10.1073/pnas.86.21.8247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kastelein R. A., Berkhout B., Overbeek G. P., van Duin J. Effect of the sequences upstream from the ribosome-binding site on the yield of protein from the cloned gene for phage MS2 coat protein. Gene. 1983 Sep;23(3):245–254. doi: 10.1016/0378-1119(83)90015-x. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Ling C. M., Hung P. P., Overby L. R. Independent assembly of Qbeta and MS2 phages in doubly infected Escherichia coli. Virology. 1970 Apr;40(4):920–929. doi: 10.1016/0042-6822(70)90138-8. [DOI] [PubMed] [Google Scholar]
  16. Ling C. M., Hung P. P., Overby L. R. Specificity in self-assembly of bacteriophages Q beta and MS2. Biochemistry. 1969 Nov;8(11):4464–4469. doi: 10.1021/bi00839a036. [DOI] [PubMed] [Google Scholar]
  17. MacWilliams M. P., Celander D. W., Gardner J. F. Direct genetic selection for a specific RNA-protein interaction. Nucleic Acids Res. 1993 Dec 11;21(24):5754–5760. doi: 10.1093/nar/21.24.5754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Matthews K. S., Cole R. D. Effects of chemical modification of f2 viral coat protein on its shell-forming activity. J Mol Biol. 1972 Mar 14;65(1):17–23. doi: 10.1016/0022-2836(72)90488-3. [DOI] [PubMed] [Google Scholar]
  19. Miller C. G., Kukral A. M., Miller J. L., Movva N. R. pepM is an essential gene in Salmonella typhimurium. J Bacteriol. 1989 Sep;171(9):5215–5217. doi: 10.1128/jb.171.9.5215-5217.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Miller C. G., Strauch K. L., Kukral A. M., Miller J. L., Wingfield P. T., Mazzei G. J., Werlen R. C., Graber P., Movva N. R. N-terminal methionine-specific peptidase in Salmonella typhimurium. Proc Natl Acad Sci U S A. 1987 May;84(9):2718–2722. doi: 10.1073/pnas.84.9.2718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Min Jou W., Haegeman G., Ysebaert M., Fiers W. Nucleotide sequence of the gene coding for the bacteriophage MS2 coat protein. Nature. 1972 May 12;237(5350):82–88. doi: 10.1038/237082a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Peabody D. S., Lim F. Complementation of RNA binding site mutations in MS2 coat protein heterodimers. Nucleic Acids Res. 1996 Jun 15;24(12):2352–2359. doi: 10.1093/nar/24.12.2352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Romaniuk P. J., Lowary P., Wu H. N., Stormo G., Uhlenbeck O. C. RNA binding site of R17 coat protein. Biochemistry. 1987 Mar 24;26(6):1563–1568. doi: 10.1021/bi00380a011. [DOI] [PubMed] [Google Scholar]
  28. Sugiyama T., Hebert R. R., Hartman K. A. Ribonucleoprotein complexes formed between bacteriophage MS2 RNA and MS2 protein in vitro. J Mol Biol. 1967 May 14;25(3):455–463. doi: 10.1016/0022-2836(67)90198-2. [DOI] [PubMed] [Google Scholar]
  29. Sugiyama T., Nakada D. Control of translation of MS2 RNA cistrons by MS2 coat protein. Proc Natl Acad Sci U S A. 1967 Jun;57(6):1744–1750. doi: 10.1073/pnas.57.6.1744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sugiyama T., Nakada D. Translational control of bacteriophage MS2 RNA cistrons by MS2 coat protein: polyacrylamide gel electrophoretic analysis of proteins synthesized in vitro. J Mol Biol. 1968 Feb 14;31(3):431–440. doi: 10.1016/0022-2836(68)90419-1. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Weber K. Amino acid sequence studies on the tryptic peptides of the coat protein of the bacteriophage R17. Biochemistry. 1967 Oct;6(10):3144–3154. doi: 10.1021/bi00862a023. [DOI] [PubMed] [Google Scholar]
  34. Witherell G. W., Gott J. M., Uhlenbeck O. C. Specific interaction between RNA phage coat proteins and RNA. Prog Nucleic Acid Res Mol Biol. 1991;40:185–220. doi: 10.1016/s0079-6603(08)60842-9. [DOI] [PubMed] [Google Scholar]
  35. de Smit M. H., van Duin J. Control of prokaryotic translational initiation by mRNA secondary structure. Prog Nucleic Acid Res Mol Biol. 1990;38:1–35. doi: 10.1016/s0079-6603(08)60707-2. [DOI] [PubMed] [Google Scholar]
  36. de Smit M. H., van Duin J. Translational initiation at the coat-protein gene of phage MS2: native upstream RNA relieves inhibition by local secondary structure. Mol Microbiol. 1993 Sep;9(5):1079–1088. doi: 10.1111/j.1365-2958.1993.tb01237.x. [DOI] [PubMed] [Google Scholar]

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