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. 1988 Jul;8(7):2737–2744. doi: 10.1128/mcb.8.7.2737

Leader length and secondary structure modulate mRNA function under conditions of stress.

M Kozak 1
PMCID: PMC363489  PMID: 3405216

Abstract

Simian virus 40-based plasmids that direct the synthesis of preproinsulin in cultured monkey cells were used to study the effects of mRNA structure on translational efficiency. Lengthening the leader sequence enhanced translation in this system. The enhancement was most obvious when an unstructured sequence (two, four, or eight copies of the oligonucleotide AGCTAAGTAAGTAAGTA) was inserted upstream from a region of deliberate secondary structure; the degree of enhancement was proportional to the number of copies of the inserted oligonucleotide. Lengthening the leader sequence on the 3' side of a stem-and-loop structure, in contrast, did not offset the potentially inhibitory effect of the hairpin structure. Both the facilitating effect of length and the inhibitory effect of secondary structure were demonstrated most easily under conditions of mRNA competition, which was brought about by an abrupt shift in the tonicity of the culture medium. These experiments suggest a simple structural basis for the long-recognized differential response of viral and cellular mRNAs to hypertonic stress. The fact that the translatability of structure-prone mRNAs varies with changes in the environment may also have general implications for gene expression in eucaryotic cells.

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Selected References

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

  1. Abraham J. A., Whang J. L., Tumolo A., Mergia A., Friedman J., Gospodarowicz D., Fiddes J. C. Human basic fibroblast growth factor: nucleotide sequence and genomic organization. EMBO J. 1986 Oct;5(10):2523–2528. doi: 10.1002/j.1460-2075.1986.tb04530.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angel P., Allegretto E. A., Okino S. T., Hattori K., Boyle W. J., Hunter T., Karin M. Oncogene jun encodes a sequence-specific trans-activator similar to AP-1. Nature. 1988 Mar 10;332(6160):166–171. doi: 10.1038/332166a0. [DOI] [PubMed] [Google Scholar]
  3. Beals C. R., Wilson C. B., Perlmutter R. M. A small multigene family encodes Gi signal-transduction proteins. Proc Natl Acad Sci U S A. 1987 Nov;84(22):7886–7890. doi: 10.1073/pnas.84.22.7886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berkner K. L., Schaffhausen B. S., Roberts T. M., Sharp P. A. Abundant expression of polyomavirus middle T antigen and dihydrofolate reductase in an adenovirus recombinant. J Virol. 1987 Apr;61(4):1213–1220. doi: 10.1128/jvi.61.4.1213-1220.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bonthron D. T., Morton C. C., Orkin S. H., Collins T. Platelet-derived growth factor A chain: gene structure, chromosomal location, and basis for alternative mRNA splicing. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1492–1496. doi: 10.1073/pnas.85.5.1492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Butnick N. Z., Miyamoto C., Chizzonite R., Cullen B. R., Ju G., Skalka A. M. Regulation of the human c-myc gene: 5' noncoding sequences do not affect translation. Mol Cell Biol. 1985 Nov;5(11):3009–3016. doi: 10.1128/mcb.5.11.3009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Damante G., Filetti S., Rapoport B. Nucleotide sequence and characterization of the 5' flanking region of the rat Ha-ras protooncogene. Proc Natl Acad Sci U S A. 1987 Feb;84(3):774–778. doi: 10.1073/pnas.84.3.774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Delli Bovi P., Curatola A. M., Kern F. G., Greco A., Ittmann M., Basilico C. An oncogene isolated by transfection of Kaposi's sarcoma DNA encodes a growth factor that is a member of the FGF family. Cell. 1987 Aug 28;50(5):729–737. doi: 10.1016/0092-8674(87)90331-x. [DOI] [PubMed] [Google Scholar]
  9. Fan H., Penman S. Regulation of protein synthesis in mammalian cells. II. Inhibition of protein synthesis at the level of initiation during mitosis. J Mol Biol. 1970 Jun 28;50(3):655–670. doi: 10.1016/0022-2836(70)90091-4. [DOI] [PubMed] [Google Scholar]
  10. Fenwick M. L., Walker M. J. Suppression of the synthesis of cellular macromolecules by herpes simplex virus. J Gen Virol. 1978 Oct;41(1):37–51. doi: 10.1099/0022-1317-41-1-37. [DOI] [PubMed] [Google Scholar]
  11. Frielle T., Collins S., Daniel K. W., Caron M. G., Lefkowitz R. J., Kobilka B. K. Cloning of the cDNA for the human beta 1-adrenergic receptor. Proc Natl Acad Sci U S A. 1987 Nov;84(22):7920–7924. doi: 10.1073/pnas.84.22.7920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gallie D. R., Sleat D. E., Watts J. W., Turner P. C., Wilson T. M. Mutational analysis of the tobacco mosaic virus 5'-leader for altered ability to enhance translation. Nucleic Acids Res. 1988 Feb 11;16(3):883–893. doi: 10.1093/nar/16.3.883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gallione C. J., Greene J. R., Iverson L. E., Rose J. K. Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus N and NS proteins. J Virol. 1981 Aug;39(2):529–535. doi: 10.1128/jvi.39.2.529-535.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Garry R. F., Ulug E. T., Bose H. R., Jr Induction of stress proteins in Sindbis virus- and vesicular stomatitis virus-infected cells. Virology. 1983 Sep;129(2):319–332. doi: 10.1016/0042-6822(83)90171-x. [DOI] [PubMed] [Google Scholar]
  15. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  16. Gronemeyer H., Turcotte B., Quirin-Stricker C., Bocquel M. T., Meyer M. E., Krozowski Z., Jeltsch J. M., Lerouge T., Garnier J. M., Chambon P. The chicken progesterone receptor: sequence, expression and functional analysis. EMBO J. 1987 Dec 20;6(13):3985–3994. doi: 10.1002/j.1460-2075.1987.tb02741.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hagag N., Lacal J. C., Graber M., Aaronson S., Viola M. V. Microinjection of ras p21 induces a rapid rise in intracellular pH. Mol Cell Biol. 1987 May;7(5):1984–1988. doi: 10.1128/mcb.7.5.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Haley J., Whittle N., Bennet P., Kinchington D., Ullrich A., Waterfield M. The human EGF receptor gene: structure of the 110 kb locus and identification of sequences regulating its transcription. Oncogene Res. 1987 Sep-Oct;1(4):375–396. [PubMed] [Google Scholar]
  19. Hickey E. D., Weber L. A. Modulation of heat-shock polypeptide synthesis in HeLa cells during hyperthermia and recovery. Biochemistry. 1982 Mar 30;21(7):1513–1521. doi: 10.1021/bi00536a008. [DOI] [PubMed] [Google Scholar]
  20. Hirai H., Maru Y., Hagiwara K., Nishida J., Takaku F. A novel putative tyrosine kinase receptor encoded by the eph gene. Science. 1987 Dec 18;238(4834):1717–1720. doi: 10.1126/science.2825356. [DOI] [PubMed] [Google Scholar]
  21. Honkawa H., Masahashi W., Hashimoto S., Hashimoto-Gotoh T. Identification of the principal promoter sequence of the c-H-ras transforming oncogene: deletion analysis of the 5'-flanking region by focus formation assay. Mol Cell Biol. 1987 Aug;7(8):2933–2940. doi: 10.1128/mcb.7.8.2933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ives H. E., Daniel T. O. Interrelationship between growth factor-induced pH changes and intracellular Ca2+. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1950–1954. doi: 10.1073/pnas.84.7.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jackson R. J. The heat-shock response in Drosophila KC 161 cells. mRNA competition is the main explanation for reduction of normal protein synthesis. Eur J Biochem. 1986 Aug 1;158(3):623–634. doi: 10.1111/j.1432-1033.1986.tb09800.x. [DOI] [PubMed] [Google Scholar]
  24. Jacobs K., Shoemaker C., Rudersdorf R., Neill S. D., Kaufman R. J., Mufson A., Seehra J., Jones S. S., Hewick R., Fritsch E. F. Isolation and characterization of genomic and cDNA clones of human erythropoietin. 1985 Feb 28-Mar 6Nature. 313(6005):806–810. doi: 10.1038/313806a0. [DOI] [PubMed] [Google Scholar]
  25. Johansen H., Schümperli D., Rosenberg M. Affecting gene expression by altering the length and sequence of the 5' leader. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7698–7702. doi: 10.1073/pnas.81.24.7698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jones D. T., Reed R. R. Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J Biol Chem. 1987 Oct 15;262(29):14241–14249. [PubMed] [Google Scholar]
  27. Karch F., Török I., Tissières A. Extensive regions of homology in front of the two hsp70 heat shock variant genes in Drosophila melanogaster. J Mol Biol. 1981 May 25;148(3):219–230. doi: 10.1016/0022-2836(81)90536-2. [DOI] [PubMed] [Google Scholar]
  28. Kaye F., Battey J., Nau M., Brooks B., Seifter E., De Greve J., Birrer M., Sausville E., Minna J. Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing. Mol Cell Biol. 1988 Jan;8(1):186–195. doi: 10.1128/mcb.8.1.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kohno K., Uchida T., Ohkubo H., Nakanishi S., Nakanishi T., Fukui T., Ohtsuka E., Ikehara M., Okada Y. Amino acid sequence of mammalian elongation factor 2 deduced from the cDNA sequence: homology with GTP-binding proteins. Proc Natl Acad Sci U S A. 1986 Jul;83(14):4978–4982. doi: 10.1073/pnas.83.14.4978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kozak M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol. 1987 Aug 20;196(4):947–950. doi: 10.1016/0022-2836(87)90418-9. [DOI] [PubMed] [Google Scholar]
  32. Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol. 1987 Oct;7(10):3438–3445. doi: 10.1128/mcb.7.10.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kozak M. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci U S A. 1986 May;83(9):2850–2854. doi: 10.1073/pnas.83.9.2850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kozak M. Mechanism of mRNA recognition by eukaryotic ribosomes during initiation of protein synthesis. Curr Top Microbiol Immunol. 1981;93:81–123. doi: 10.1007/978-3-642-68123-3_5. [DOI] [PubMed] [Google Scholar]
  35. Kozak M. Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo. Nature. 1984 Mar 15;308(5956):241–246. doi: 10.1038/308241a0. [DOI] [PubMed] [Google Scholar]
  36. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  37. Kozak M. Regulation of protein synthesis in virus-infected animal cells. Adv Virus Res. 1986;31:229–292. doi: 10.1016/S0065-3527(08)60265-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kruppa J., Clemens M. J. Differential kinetics of changes in the state of phosphorylation of ribosomal protein S6 and in the rate of protein synthesis in MPC 11 cells during tonicity shifts. EMBO J. 1984 Jan;3(1):95–100. doi: 10.1002/j.1460-2075.1984.tb01767.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lannigan D. A., Knauf P. A., Macara I. G. Relationship of the decreases in protein synthesis and intracellular Na+ during friend murine erythroleukemic cell differentiation. J Biol Chem. 1986 Nov 5;261(31):14430–14436. [PubMed] [Google Scholar]
  40. Logan J., Shenk T. Adenovirus tripartite leader sequence enhances translation of mRNAs late after infection. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3655–3659. doi: 10.1073/pnas.81.12.3655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Lomedico P. T., McAndrew S. J. Eukaryotic ribosomes can recognize preproinsulin initiation codons irrespective of their position relative to the 5' end of mRNA. Nature. 1982 Sep 16;299(5880):221–226. doi: 10.1038/299221a0. [DOI] [PubMed] [Google Scholar]
  42. Martin D., Jr, Tomkins G. M., Granner D. Synthesis and induction of tyrosine aminotransferase in synchronized hepatoma cells in culture. Proc Natl Acad Sci U S A. 1969 Jan;62(1):248–255. doi: 10.1073/pnas.62.1.248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. McGarry T. J., Lindquist S. The preferential translation of Drosophila hsp70 mRNA requires sequences in the untranslated leader. Cell. 1985 Oct;42(3):903–911. doi: 10.1016/0092-8674(85)90286-7. [DOI] [PubMed] [Google Scholar]
  44. Morgan D. O., Edman J. C., Standring D. N., Fried V. A., Smith M. C., Roth R. A., Rutter W. J. Insulin-like growth factor II receptor as a multifunctional binding protein. Nature. 1987 Sep 24;329(6137):301–307. doi: 10.1038/329301a0. [DOI] [PubMed] [Google Scholar]
  45. Muñoz A., Alonso M. A., Carrasco L. Synthesis of heat-shock proteins in HeLa cells: inhibition by virus infection. Virology. 1984 Aug;137(1):150–159. doi: 10.1016/0042-6822(84)90018-7. [DOI] [PubMed] [Google Scholar]
  46. Nuss D. L., Koch G. Differential inhibition of vesicular stomatitis virus polypeptide synthesis by hypertonic initiation block. J Virol. 1975 Jan;17(1):283–286. doi: 10.1128/jvi.17.1.283-286.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Nuss D. L., Koch G. Variation in the relative synthesis of immunoglobulin G and non-immunoglobulin G proteins in cultured MPC-11 cells with changes in the overall rate of polypeptide chain initiation and elongation. J Mol Biol. 1976 Apr 15;102(3):601–612. doi: 10.1016/0022-2836(76)90337-5. [DOI] [PubMed] [Google Scholar]
  48. Nuss D. L., Oppermann H., Koch G. Selective blockage of initiation of host protein synthesis in RNA-virus-infected cells. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1258–1262. doi: 10.1073/pnas.72.4.1258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Ohno S., Kawasaki H., Imajoh S., Suzuki K., Inagaki M., Yokokura H., Sakoh T., Hidaka H. Tissue-specific expression of three distinct types of rabbit protein kinase C. Nature. 1987 Jan 8;325(7000):161–166. doi: 10.1038/325161a0. [DOI] [PubMed] [Google Scholar]
  50. Papanicolaou C., Lecomte P., Ninio J. Mnemonic aspects of Escherichia coli DNA polymerase I. Interaction with one template influences the next interaction with another template. J Mol Biol. 1986 Jun 5;189(3):435–448. doi: 10.1016/0022-2836(86)90315-3. [DOI] [PubMed] [Google Scholar]
  51. Pawlowski P. J. Differential effect of hypertonic initiation block on the synthesis of collagen chains by cultured chick embryo cells. Biochemistry. 1982 Jan 5;21(1):34–38. doi: 10.1021/bi00530a006. [DOI] [PubMed] [Google Scholar]
  52. Rao C. D., Igarashi H., Chiu I. M., Robbins K. C., Aaronson S. A. Structure and sequence of the human c-sis/platelet-derived growth factor 2 (SIS/PDGF2) transcriptional unit. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2392–2396. doi: 10.1073/pnas.83.8.2392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Rebagliati M. R., Melton D. A. Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity. Cell. 1987 Feb 27;48(4):599–605. doi: 10.1016/0092-8674(87)90238-8. [DOI] [PubMed] [Google Scholar]
  54. Saborio J. L., Pong S. S., Koch G. Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. J Mol Biol. 1974 May 15;85(2):195–211. doi: 10.1016/0022-2836(74)90360-x. [DOI] [PubMed] [Google Scholar]
  55. Scott M. P., Pardue M. L. Translational control in lysates of Drosophila melanogaster cells. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3353–3357. doi: 10.1073/pnas.78.6.3353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Selten G., Cuypers H. T., Boelens W., Robanus-Maandag E., Verbeek J., Domen J., van Beveren C., Berns A. The primary structure of the putative oncogene pim-1 shows extensive homology with protein kinases. Cell. 1986 Aug 15;46(4):603–611. doi: 10.1016/0092-8674(86)90886-x. [DOI] [PubMed] [Google Scholar]
  57. Stanton L. W., Bishop J. M. Alternative processing of RNA transcribed from NMYC. Mol Cell Biol. 1987 Dec;7(12):4266–4272. doi: 10.1128/mcb.7.12.4266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tal M., King C. R., Kraus M. H., Ullrich A., Schlessinger J., Givol D. Human HER2 (neu) promoter: evidence for multiple mechanisms for transcriptional initiation. Mol Cell Biol. 1987 Jul;7(7):2597–2601. doi: 10.1128/mcb.7.7.2597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Tamkun J. W., DeSimone D. W., Fonda D., Patel R. S., Buck C., Horwitz A. F., Hynes R. O. Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin. Cell. 1986 Jul 18;46(2):271–282. doi: 10.1016/0092-8674(86)90744-0. [DOI] [PubMed] [Google Scholar]
  61. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  62. Wagner R. W., Nishikura K. Cell cycle expression of RNA duplex unwindase activity in mammalian cells. Mol Cell Biol. 1988 Feb;8(2):770–777. doi: 10.1128/mcb.8.2.770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]
  64. Yamanashi Y., Fukushige S., Semba K., Sukegawa J., Miyajima N., Matsubara K., Yamamoto T., Toyoshima K. The yes-related cellular gene lyn encodes a possible tyrosine kinase similar to p56lck. Mol Cell Biol. 1987 Jan;7(1):237–243. doi: 10.1128/mcb.7.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Yates J. R., Nuss D. L. Resistance to inhibitors of mammalian cell protein synthesis induced by preincubation in hypertonic growth medium. J Biol Chem. 1982 Dec 25;257(24):15030–15034. [PubMed] [Google Scholar]
  66. Yates J. R., Nuss D. L. Time-dependent increase in the resistance of mammalian cell protein synthesis to inhibition by hypertonic medium. J Biol Chem. 1982 Apr 10;257(7):3458–3461. [PubMed] [Google Scholar]
  67. Yoshida T., Miyagawa K., Odagiri H., Sakamoto H., Little P. F., Terada M., Sugimura T. Genomic sequence of hst, a transforming gene encoding a protein homologous to fibroblast growth factors and the int-2-encoded protein. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7305–7309. doi: 10.1073/pnas.84.20.7305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. van Ormondt H., Galibert F. Nucleotide sequences of adenovirus DNAs. Curr Top Microbiol Immunol. 1984;110:73–142. doi: 10.1007/978-3-642-46494-2_4. [DOI] [PubMed] [Google Scholar]
  69. van der Valk J., Verlaan I., de Laat S. W., Moolenaar W. H. Expression of pp60v-src alters the ionic permeability of the plasma membrane in rat cells. J Biol Chem. 1987 Feb 25;262(6):2431–2434. [PubMed] [Google Scholar]

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