Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1973 Sep;70(9):2605–2609. doi: 10.1073/pnas.70.9.2605

Discriminatory Ribosome Rebinding of Isolated Regions of Protein Synthesis Initiation from the Ribonucleic Acid of Bacteriophage R17

Joan Argetsinger Steitz 1
PMCID: PMC427065  PMID: 4582190

Abstract

To determine whether bacterial ribosomes recognize a distinguishing feature in the immediate vicinity of actual initiator codons or are directed to these sites through involvement of other portion(s) of the mRNA molecule, the interaction between ribosomes and defined 32P-labeled initiator fragments from R17 RNA was studied. When incubated with mixtures of the three sites, ribosomes from Bacillus stearothermophilus (which initiate only the A protein on intact phage RNA) are able to select out the A fragment and discriminate against the coat and replicase initiator regions. By contrast, Escherichia coli ribosomes do not rebind that coat-protein region of R17 most efficiently, as they in the native RNA, but likewise prefer the A-protein initiator fragment. In both cases, ribosome binding of the isolated A site is comparable by several criteria to normal polypeptide-chain initiation on an intact R17 messenger RNA in vitro. E. coli ribosomal preference for the A site is confirmed in experiments with randomly fragmented R17 RNA, by both the initiation dipeptide and ribosome protection assay. Thus the A-protein ribosome-binding site of R17 RNA appears intrinsically to be a good initiator, while efficient recognition of the coat and replicase regions requires the participation of some portion of the remainder of the phage RNA molecule.

Keywords: messenger RNA, specificity in polypeptide chain initiation, RNA structure, E. coli ribosomes, B. stearothermophilus ribosomes

Full text

PDF
2605

Images in this article

2606Image
on p.2606
2606Image
on p.2606

Selected References

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

  1. Adams J. M., Cory S., Spahr P. F. Nucleotide sequences of fragments of R17 bacteriophage RNA from the region immediately preceding the coat-protein cistron. Eur J Biochem. 1972 Sep 25;29(3):469–479. doi: 10.1111/j.1432-1033.1972.tb02011.x. [DOI] [PubMed] [Google Scholar]
  2. Adams J. M., Spahr P. F., Cory S. Nucleotide sequence from the 5' end to the first cistron of R17 bacteriophage ribonucleic acid. Biochemistry. 1972 Mar 14;11(6):976–988. doi: 10.1021/bi00756a006. [DOI] [PubMed] [Google Scholar]
  3. Bretscher M. S. Direct translation of bacteriophage fd DNA in the absence of neomycin B. J Mol Biol. 1969 Jun 28;42(3):595–598. doi: 10.1016/0022-2836(69)90247-2. [DOI] [PubMed] [Google Scholar]
  4. Fukami H., Imahori K. Control of translation by the conformation of messenger RNA. Proc Natl Acad Sci U S A. 1971 Mar;68(3):570–573. doi: 10.1073/pnas.68.3.570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gesteland R. F., Spahr P. F. Translation of R-17 RNA fragments. Cold Spring Harb Symp Quant Biol. 1969;34:707–716. doi: 10.1101/sqb.1969.034.01.080. [DOI] [PubMed] [Google Scholar]
  6. Gralla J., Crothers D. M. Free energy of imperfect nucleic acid helices. II. Small hairpin loops. J Mol Biol. 1973 Feb 5;73(4):497–511. doi: 10.1016/0022-2836(73)90096-x. [DOI] [PubMed] [Google Scholar]
  7. Groner Y., Pollack Y., Berissi H., Revel M. Cistron specific translation control protein in Escherichia coli. Nat New Biol. 1972 Sep 6;239(88):16–19. doi: 10.1038/newbio239016a0. [DOI] [PubMed] [Google Scholar]
  8. Gupta S. L., Chen J., Schaefer L., Lengyel P., Weissman S. M. Nucleotide sequence of a ribosome attachment site of bacteriophage f2 RNA. Biochem Biophys Res Commun. 1970 Jun 5;39(5):883–888. doi: 10.1016/0006-291x(70)90406-7. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Jeppesen P. G., Steitz J. A., Gesteland R. F., Spahr P. F. Gene order in the bacteriophage R17 RNA: 5'-a protein-coat protein-synthetase-3'. Nature. 1970 Apr 18;226(5242):230–237. doi: 10.1038/226230a0. [DOI] [PubMed] [Google Scholar]
  11. Kozak M., Nathans D. Differential inhibition of coliphage MS2 protein synthesis by ribosome-directed antibiotics. J Mol Biol. 1972 Sep 14;70(1):41–55. doi: 10.1016/0022-2836(72)90162-3. [DOI] [PubMed] [Google Scholar]
  12. Lee-Huang S., Ochoa S. Messenger discriminating species of initiation factor F3. Nat New Biol. 1971 Dec 22;234(51):236–239. doi: 10.1038/newbio234236a0. [DOI] [PubMed] [Google Scholar]
  13. Lee-Huang S., Ochoa S. Specific inhibitors of MS2 and late T4 RNA translation in E. coli. Biochem Biophys Res Commun. 1972 Oct 17;49(2):371–376. doi: 10.1016/0006-291x(72)90420-2. [DOI] [PubMed] [Google Scholar]
  14. Lodish H. F. Bacteriophage f2 RNA: control of translation and gene order. Nature. 1968 Oct 26;220(5165):345–350. doi: 10.1038/220345a0. [DOI] [PubMed] [Google Scholar]
  15. Lodish H. F. Independent initiation of translation of two bacteriophage f2 proteins. Biochem Biophys Res Commun. 1969 Sep 24;37(1):127–136. doi: 10.1016/0006-291x(69)90890-0. [DOI] [PubMed] [Google Scholar]
  16. Lodish H. F. Independent translation of the genes of bacteriophage f2 RNA. J Mol Biol. 1968 Mar 28;32(3):681–685. doi: 10.1016/0022-2836(68)90351-3. [DOI] [PubMed] [Google Scholar]
  17. Lodish H. F. Secondary structure of bacteriophage f2 ribonucleic acid and the initiation of in vitro protein biosynthesis. J Mol Biol. 1970 Jun 28;50(3):689–702. doi: 10.1016/0022-2836(70)90093-8. [DOI] [PubMed] [Google Scholar]
  18. Lodish H. F. Species specificity of polypeptide chain initiation. Nature. 1969 Nov 29;224(5222):867–870. doi: 10.1038/224867a0. [DOI] [PubMed] [Google Scholar]
  19. Lodish H. F. Thermal melting of bacteriophage f2 RNA and initiation of synthesis of the maturation protein. J Mol Biol. 1971 Mar 28;56(3):627–632. doi: 10.1016/0022-2836(71)90406-2. [DOI] [PubMed] [Google Scholar]
  20. Lucas-Lenard J. Protein biosynthesis. Annu Rev Biochem. 1971;40:409–448. doi: 10.1146/annurev.bi.40.070171.002205. [DOI] [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. Pollack Y., Groner Y., Aviv(Greenshpan) H., Revel M. Role of initiation factor B (F3) in the preferential translation of T4 late messenger RNA in T4 infected E. Coli. FEBS Lett. 1970 Aug 17;9(4):218–221. doi: 10.1016/0014-5793(70)80359-3. [DOI] [PubMed] [Google Scholar]
  23. Revel M., Aviv(Greenshpan) H., Groner Y., Pollack Y. Fractionation of translation initiation factor B (F3) into cistron-specific species. FEBS Lett. 1970 Aug 17;9(4):213–217. doi: 10.1016/0014-5793(70)80358-1. [DOI] [PubMed] [Google Scholar]
  24. Robertson H. D., Barrell B. G., Weith H. L., Donelson J. E. Isolation and sequence analysis of a ribosome-protected fragment from bacteriophage phiX 174 DNA. Nat New Biol. 1973 Jan 10;241(106):38–40. doi: 10.1038/newbio241038a0. [DOI] [PubMed] [Google Scholar]
  25. Robertson H. D., Lodish H. F. Messenger characteristics of nascent bacteriophage RNA. Proc Natl Acad Sci U S A. 1970 Oct;67(2):710–716. doi: 10.1073/pnas.67.2.710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Robertson H. D., Webster R. E., Zinder N. D. Purification and properties of ribonuclease III from Escherichia coli. J Biol Chem. 1968 Jan 10;243(1):82–91. [PubMed] [Google Scholar]
  27. Roufa D. J., Leder P. Biosynthesis of phage-specific initiation dipeptides. A purified biosynthetic system derived from Escherichia coli. J Biol Chem. 1971 May 25;246(10):3160–3167. [PubMed] [Google Scholar]
  28. Shimura Y., Kaizer H., Nathans D. Fragments of MS2 RNA as messengers for specific bacteriophage proteins: fragments from fluorouracil-containing particles. J Mol Biol. 1968 Dec;38(3):453–455. doi: 10.1016/0022-2836(68)90402-6. [DOI] [PubMed] [Google Scholar]
  29. Staples D. H., Hindley J., Billeter M. A., Weissmann C. Localization of Q-beta maturation cistron ribosome binding site. Nat New Biol. 1971 Sep 15;234(50):202–204. doi: 10.1038/newbio234202a0. [DOI] [PubMed] [Google Scholar]
  30. Staples D. H., Hindley J. Ribosome binding site of Q-beta RNA polymerase cistron. Nat New Biol. 1971 Sep 15;234(50):211–212. doi: 10.1038/newbio234211a0. [DOI] [PubMed] [Google Scholar]
  31. Steitz J. A., Dube S. K., Rudland P. S. Control of translation of T4 phage: altered ribosome binding at R17 initiation sites. Nature. 1970 May 30;226(5248):824–827. doi: 10.1038/226824a0. [DOI] [PubMed] [Google Scholar]
  32. Steitz J. A. Oligonucleotide sequence of replicase initiation site in Q RNA. Nat New Biol. 1972 Mar 22;236(64):71–75. doi: 10.1038/newbio236071a0. [DOI] [PubMed] [Google Scholar]
  33. Steitz J. A. Polypeptide chain initiation: nucleotide sequences of the three ribosomal binding sites in bacteriophage R17 RNA. Nature. 1969 Dec 6;224(5223):957–964. doi: 10.1038/224957a0. [DOI] [PubMed] [Google Scholar]
  34. Steitz J. A. Specific recognition of non-initiator regions in RNA bacteriophage messengers by ribosomes of Bacillus stearothermophilus. J Mol Biol. 1973 Jan;73(1):1–16. doi: 10.1016/0022-2836(73)90155-1. [DOI] [PubMed] [Google Scholar]
  35. Thach S., Boedtker H. High molecular weight fragments of R17 bacteriophage RNA produced by pancreatic ribonuclease. J Mol Biol. 1969 Nov 14;45(3):451–465. doi: 10.1016/0022-2836(69)90305-2. [DOI] [PubMed] [Google Scholar]
  36. Voorma H. O., Benne R., den Hertog T. J. Binding of aminoacyl-tRNA to ribosomes programmed with bacteriophage MS2-RNA. Eur J Biochem. 1971 Feb;18(4):451–462. doi: 10.1111/j.1432-1033.1971.tb01263.x. [DOI] [PubMed] [Google Scholar]
  37. Voynow P., Kurland C. G. Stoichiometry of the 30S ribosomal proteins of Escherichia coli. Biochemistry. 1971 Feb 2;10(3):517–524. doi: 10.1021/bi00779a026. [DOI] [PubMed] [Google Scholar]
  38. Weber H., Billeter M. A., Kahane S., Weissmann C., Hindley J., Porter A. Molecular basis for repressor activity of Q replicase. Nat New Biol. 1972 Jun 7;237(75):166–170. doi: 10.1038/newbio237166a0. [DOI] [PubMed] [Google Scholar]
  39. de Wachter R., Merregaert J., Vandenberghe A., Contreras R., Fiers W. Studies on the bacteriophage MS2. The untranslated 5'-terminal nucleotide sequence preceding the first cistron. Eur J Biochem. 1971 Oct 14;22(3):400–414. doi: 10.1111/j.1432-1033.1971.tb01558.x. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES