Abstract
We present the results of in vitro binding studies aimed at defining the key recognition elements on the MS2 RNA translational operator (TR) essential for complex formation with coat protein. We have used chemically synthesized operators carrying modified functional groups at defined nucleotide positions, which are essential for recognition by the phage coat protein. These experiments have been complemented with modification-binding interference assays. The results confirm that the complexes which form between TR and RNA-free phage capsids, the X-ray structure of which has recently been reported at 3.0 A, are identical to those which form in solution between TR and a single coat protein dimer. There are also effects on operator affinity which cannot be explained simply by the alteration of direct RNA-protein contacts and may reflect changes in the conformational equilibrium of the unliganded operator. The results also provide support for the approach of using modified oligoribonucleotides to investigate the details of RNA-ligand interactions.
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- 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]
- Carey J., Uhlenbeck O. C. Kinetic and thermodynamic characterization of the R17 coat protein-ribonucleic acid interaction. Biochemistry. 1983 May 24;22(11):2610–2615. doi: 10.1021/bi00280a003. [DOI] [PubMed] [Google Scholar]
- Donis-Keller H., Maxam A. M., Gilbert W. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977 Aug;4(8):2527–2538. doi: 10.1093/nar/4.8.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ehresmann C., Baudin F., Mougel M., Romby P., Ebel J. P., Ehresmann B. Probing the structure of RNAs in solution. Nucleic Acids Res. 1987 Nov 25;15(22):9109–9128. doi: 10.1093/nar/15.22.9109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Haneef I., Talbot S. J., Stockley P. G. Modeling loop structures in proteins and nucleic acids: an RNA stem-loop. J Mol Graph. 1989 Dec;7(4):186–195. doi: 10.1016/0263-7855(89)80001-3. [DOI] [PubMed] [Google Scholar]
- Mastico R. A., Talbot S. J., Stockley P. G. Multiple presentation of foreign peptides on the surface of an RNA-free spherical bacteriophage capsid. J Gen Virol. 1993 Apr;74(Pt 4):541–548. doi: 10.1099/0022-1317-74-4-541. [DOI] [PubMed] [Google Scholar]
- Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Murray J. B., Collier A. K., Arnold J. R. A general purification procedure for chemically synthesized oligoribonucleotides. Anal Biochem. 1994 Apr;218(1):177–184. doi: 10.1006/abio.1994.1157. [DOI] [PubMed] [Google Scholar]
- 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]
- Peabody D. S. Translational repression by bacteriophage MS2 coat protein does not require cysteine residues. Nucleic Acids Res. 1989 Aug 11;17(15):6017–6027. doi: 10.1093/nar/17.15.6017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Romaniuk P. J., Uhlenbeck O. C. Nucleoside and nucleotide inactivation of R17 coat protein: evidence for a transient covalent RNA-protein bond. Biochemistry. 1985 Jul 16;24(15):4239–4244. doi: 10.1021/bi00336a064. [DOI] [PubMed] [Google Scholar]
- Sanger F., Coulson A. R. The use of thin acrylamide gels for DNA sequencing. FEBS Lett. 1978 Mar 1;87(1):107–110. doi: 10.1016/0014-5793(78)80145-8. [DOI] [PubMed] [Google Scholar]
- 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]
- Sorger P. K., Stockley P. G., Harrison S. C. Structure and assembly of turnip crinkle virus. II. Mechanism of reassembly in vitro. J Mol Biol. 1986 Oct 20;191(4):639–658. doi: 10.1016/0022-2836(86)90451-1. [DOI] [PubMed] [Google Scholar]
- Stockley P. G., Stonehouse N. J., Walton C., Walters D. A., Medina G., Macedo J. M., Hill H. R., Goodman S. T., Talbot S. J., Tewary H. K. Molecular mechanism of RNA-phage morphogenesis. Biochem Soc Trans. 1993 Aug;21(3):627–633. doi: 10.1042/bst0210627. [DOI] [PubMed] [Google Scholar]
- 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]
- Talbot S. J., Medina G., Fishwick C. W., Haneef I., Stockley P. G. Hyperreactivity of adenines and conformational flexibility of a translational repression site. FEBS Lett. 1991 May 20;283(1):159–164. doi: 10.1016/0014-5793(91)80576-o. [DOI] [PubMed] [Google Scholar]
- Uhlenbeck O. C., Carey J., Romaniuk P. J., Lowary P. T., Beckett D. Interaction of R17 coat protein with its RNA binding site for translational repression. J Biomol Struct Dyn. 1983 Oct;1(2):539–552. doi: 10.1080/07391102.1983.10507460. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]