Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1988 Nov;7(11):3559–3569. doi: 10.1002/j.1460-2075.1988.tb03233.x

Ribosome pausing and stacking during translation of a eukaryotic mRNA.

S L Wolin 1, P Walter 1
PMCID: PMC454858  PMID: 2850168

Abstract

We have devised a sensitive assay to determine the distribution of translating ribosomes on a mRNA. Using this assay to monitor ribosome transit on bovine preprolactin mRNA, we have detected four major positions of ribosome pausing in both wheat-germ and rabbit reticulocyte extracts. Two of these rate-limiting steps represent initiation and termination. One pause occurs after approximately 75 amino acids have been polymerized; signal recognition particle arrests preprolactin synthesis at this position. The other internal pause occurs at 160 amino acids. In these latter two cases ribosomes stop at a GGC glycine codon; however, two other GGC codons are translated without apparent pausing. Surprisingly, we find that up to nine ribosomes are tightly stacked behind each pausing ribosome, such that the ribosome centers are only 27-29 nucleotides apart. The assay should prove useful for probing mechanisms of translational regulation.

Full text

PDF
3559

Images in this article

3561Image
on p.3561
3562Image
on p.3562
3563Image
on p.3563
3564Image
on p.3564
3565Image
on p.3565

Selected References

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

  1. Alberts B. M. The DNA enzymology of protein machines. Cold Spring Harb Symp Quant Biol. 1984;49:1–12. doi: 10.1101/sqb.1984.049.01.003. [DOI] [PubMed] [Google Scholar]
  2. Bergmann J. E., Lodish H. F. A kinetic model of protein synthesis. Application to hemoglobin synthesis and translational control. J Biol Chem. 1979 Dec 10;254(23):11927–11937. [PubMed] [Google Scholar]
  3. Both G. W., Furuichi Y., Muthukrishnan S., Shatkin A. J. Effect of 5'-terminal structure and base composition on polyribonucleotide binding to ribosomes. J Mol Biol. 1976 Jul 5;104(3):637–658. doi: 10.1016/0022-2836(76)90126-1. [DOI] [PubMed] [Google Scholar]
  4. Cech T. R., Tanner N. K., Tinoco I., Jr, Weir B. R., Zuker M., Perlman P. S. Secondary structure of the Tetrahymena ribosomal RNA intervening sequence: structural homology with fungal mitochondrial intervening sequences. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3903–3907. doi: 10.1073/pnas.80.13.3903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Christensen A. K., Kahn L. E., Bourne C. M. Circular polysomes predominate on the rough endoplasmic reticulum of somatotropes and mammotropes in the rat anterior pituitary. Am J Anat. 1987 Jan;178(1):1–10. doi: 10.1002/aja.1001780102. [DOI] [PubMed] [Google Scholar]
  6. Colombo B., Felicetti L., Baglioni C. Inhibition of protein synthesis in reticulocytes by antibiotics. I. Effects on polysomes. Biochim Biophys Acta. 1966 Apr 18;119(1):109–119. doi: 10.1016/0005-2787(66)90043-8. [DOI] [PubMed] [Google Scholar]
  7. Dahlberg A. E., Lund E., Kjeldgaard N. O. Some effects of antibiotics on bacterial polyribosomes as studied by gel electrophoresis. J Mol Biol. 1973 Aug 25;78(4):627–636. doi: 10.1016/0022-2836(73)90284-2. [DOI] [PubMed] [Google Scholar]
  8. Erickson A. H., Blobel G. Cell-free translation of messenger RNA in a wheat germ system. Methods Enzymol. 1983;96:38–50. doi: 10.1016/s0076-6879(83)96007-x. [DOI] [PubMed] [Google Scholar]
  9. Gold L. Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem. 1988;57:199–233. doi: 10.1146/annurev.bi.57.070188.001215. [DOI] [PubMed] [Google Scholar]
  10. Haeuptle M. T., Frank R., Dobberstein B. Translation arrest by oligodeoxynucleotides complementary to mRNA coding sequences yields polypeptides of predetermined length. Nucleic Acids Res. 1986 Feb 11;14(3):1427–1448. doi: 10.1093/nar/14.3.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Heinrich R., Rapoport T. A. Mathematical modelling of translation of mRNA in eucaryotes; steady state, time-dependent processes and application to reticulocytes. J Theor Biol. 1980 Sep 21;86(2):279–313. doi: 10.1016/0022-5193(80)90008-9. [DOI] [PubMed] [Google Scholar]
  12. Hickey E. D., Weber L. A., Baglioni C. Inhibition of initiation of protein synthesis by 7-methylguanosine-5'-monophosphate. Proc Natl Acad Sci U S A. 1976 Jan;73(1):19–23. doi: 10.1073/pnas.73.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Hortsch M., Meyer D. I. Pushing the signal hypothesis: what are the limits? Biol Cell. 1984;52(1 Pt A):1–8. doi: 10.1111/j.1768-322x.1985.tb00319.x. [DOI] [PubMed] [Google Scholar]
  15. Hu M. C., Davidson N. Mapping transcription start points on cloned genomic DNA with T4 DNA polymerase: a precise and convenient technique. Gene. 1986;42(1):21–29. doi: 10.1016/0378-1119(86)90146-0. [DOI] [PubMed] [Google Scholar]
  16. Ikemura T., Ozeki H. Codon usage and transfer RNA contents: organism-specific codon-choice patterns in reference to the isoacceptor contents. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):1087–1097. doi: 10.1101/sqb.1983.047.01.123. [DOI] [PubMed] [Google Scholar]
  17. Jackson R. J., Hunt T. Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Methods Enzymol. 1983;96:50–74. doi: 10.1016/s0076-6879(83)96008-1. [DOI] [PubMed] [Google Scholar]
  18. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kozak M., Shatkin A. J. Migration of 40 S ribosomal subunits on messenger RNA in the presence of edeine. J Biol Chem. 1978 Sep 25;253(18):6568–6577. [PubMed] [Google Scholar]
  20. Kozak M., Shatkin A. J. Sequences and properties of two ribosome binding sites from the small size class of reovirus messenger RNA. J Biol Chem. 1977 Oct 10;252(19):6895–6908. [PubMed] [Google Scholar]
  21. Kozak M., Shatkin A. J. Sequences of two 5'-terminal ribosome-protected fragments from reovirus messenger RNAs. J Mol Biol. 1977 May 5;112(1):75–96. doi: 10.1016/s0022-2836(77)80157-5. [DOI] [PubMed] [Google Scholar]
  22. Lazarowitz S. G., Robertson H. D. Initiator regions from the small size class of reovirus messenger RNA protected by rabbit reticulocyte ribosomes. J Biol Chem. 1977 Nov 10;252(21):7842–7849. [PubMed] [Google Scholar]
  23. Legon S. Characterization of the ribosome-protected regions of 125I-labelled rabbit globin messenger RNA. J Mol Biol. 1976 Sep 5;106(1):37–53. doi: 10.1016/0022-2836(76)90299-0. [DOI] [PubMed] [Google Scholar]
  24. Legon S., Model P., Robertson H. D. Interaction of rabbit reticulocyte ribosomes with bacteriophage f1 mRNA and of Escherichia coli ribosomes with rabbit globin mRNA. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2692–2696. doi: 10.1073/pnas.74.7.2692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Legon S., Robertson H. D. The binding of 125I-labelled rabbit globin messenger RNA to reticulocyte ribosomes. J Mol Biol. 1976 Sep 5;106(1):23–36. doi: 10.1016/0022-2836(76)90298-9. [DOI] [PubMed] [Google Scholar]
  26. Lodish H. F., Jacobsen M. Regulation of hemoglobin synthesis. Equal rates of translation and termination of - and -globin chains. J Biol Chem. 1972 Jun 10;247(11):3622–3629. [PubMed] [Google Scholar]
  27. 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]
  28. Meyer D. I., Krause E., Dobberstein B. Secretory protein translocation across membranes-the role of the "docking protein'. Nature. 1982 Jun 24;297(5868):647–650. doi: 10.1038/297647a0. [DOI] [PubMed] [Google Scholar]
  29. Moldave K. Eukaryotic protein synthesis. Annu Rev Biochem. 1985;54:1109–1149. doi: 10.1146/annurev.bi.54.070185.005333. [DOI] [PubMed] [Google Scholar]
  30. Mueller P. P., Hinnebusch A. G. Multiple upstream AUG codons mediate translational control of GCN4. Cell. 1986 Apr 25;45(2):201–207. doi: 10.1016/0092-8674(86)90384-3. [DOI] [PubMed] [Google Scholar]
  31. Nossal N. G. Prokaryotic DNA replication systems. Annu Rev Biochem. 1983;52:581–615. doi: 10.1146/annurev.bi.52.070183.003053. [DOI] [PubMed] [Google Scholar]
  32. Protzel A., Morris A. J. Gel chromatographic analysis of nascent globin chains. Evidence of nonuniform size distribution. J Biol Chem. 1974 Jul 25;249(14):4594–4600. [PubMed] [Google Scholar]
  33. Rapoport T. A., Heinrich R., Walter P., Schulmeister T. Mathematical modeling of the effects of the signal recognition particle on translation and translocation of proteins across the endoplasmic reticulum membrane. J Mol Biol. 1987 Jun 5;195(3):621–636. doi: 10.1016/0022-2836(87)90186-0. [DOI] [PubMed] [Google Scholar]
  34. Safer B., Kemper W., Jagus R. Identification of a 48 S preinitiation complex in reticulocyte lysate. J Biol Chem. 1978 May 25;253(10):3384–3386. [PubMed] [Google Scholar]
  35. Sasavage N. L., Nilson J. H., Horowitz S., Rottman F. M. Nucleotide sequence of bovine prolactin messenger RNA. Evidence for sequence polymorphism. J Biol Chem. 1982 Jan 25;257(2):678–681. [PubMed] [Google Scholar]
  36. Siegel V., Walter P. Each of the activities of signal recognition particle (SRP) is contained within a distinct domain: analysis of biochemical mutants of SRP. Cell. 1988 Jan 15;52(1):39–49. doi: 10.1016/0092-8674(88)90529-6. [DOI] [PubMed] [Google Scholar]
  37. Siegel V., Walter P. The affinity of signal recognition particle for presecretory proteins is dependent on nascent chain length. EMBO J. 1988 Jun;7(6):1769–1775. doi: 10.1002/j.1460-2075.1988.tb03007.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Tzamarias D., Alexandraki D., Thireos G. Multiple cis-acting elements modulate the translational efficiency of GCN4 mRNA in yeast. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4849–4853. doi: 10.1073/pnas.83.13.4849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Varenne S., Buc J., Lloubes R., Lazdunski C. Translation is a non-uniform process. Effect of tRNA availability on the rate of elongation of nascent polypeptide chains. J Mol Biol. 1984 Dec 15;180(3):549–576. doi: 10.1016/0022-2836(84)90027-5. [DOI] [PubMed] [Google Scholar]
  41. Walter P., Blobel G. Signal recognition particle: a ribonucleoprotein required for cotranslational translocation of proteins, isolation and properties. Methods Enzymol. 1983;96:682–691. doi: 10.1016/s0076-6879(83)96057-3. [DOI] [PubMed] [Google Scholar]
  42. Walter P., Blobel G. Translocation of proteins across the endoplasmic reticulum III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J Cell Biol. 1981 Nov;91(2 Pt 1):557–561. doi: 10.1083/jcb.91.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Walter P., Lingappa V. R. Mechanism of protein translocation across the endoplasmic reticulum membrane. Annu Rev Cell Biol. 1986;2:499–516. doi: 10.1146/annurev.cb.02.110186.002435. [DOI] [PubMed] [Google Scholar]
  44. Werner M., Feller A., Messenguy F., Piérard A. The leader peptide of yeast gene CPA1 is essential for the translational repression of its expression. Cell. 1987 Jun 19;49(6):805–813. doi: 10.1016/0092-8674(87)90618-0. [DOI] [PubMed] [Google Scholar]
  45. Yarus M., Folley L. S. Sense codons are found in specific contexts. J Mol Biol. 1985 Apr 20;182(4):529–540. doi: 10.1016/0022-2836(85)90239-6. [DOI] [PubMed] [Google Scholar]
  46. von Heijne G., Nilsson L., Blomberg C. Models for mRNA translation: theory versus experiment. Eur J Biochem. 1978 Dec;92(2):397–402. doi: 10.1111/j.1432-1033.1978.tb12759.x. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES