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Plant Cell. Oct 1998; 10(10): 1733–1746.
PMCID: PMC143946

Inefficient reinitiation is responsible for upstream open reading frame-mediated translational repression of the maize R gene.


Maize R genes encode a small family of transcriptional activators of several structural genes in the anthocyanin biosynthetic pathway. The 5' leader region of most R genes contains a 38-codon upstream open reading frame (uORF) that previously was shown to be responsible for the repression of downstream gene expression in a transient transformation assay. In this study, we report that the 5' leader also can repress translation of the downstream luciferase gene both in the rabbit reticulocyte translation system and in transgenic rice plants. The ability to visualize the uORF peptide after in vitro translation permits quantification of both products of dicistronic mRNAs. Similarly, the construction of transgenic rice plants expressing wild-type and mutant constructs permits the quantification and correlation of steady state mRNA levels and reporter gene activities. Using these assays, we demonstrate directly that translation of the uORF is required for repression, that increasing translation of the uORF peptide decreases downstream gene expression, and that repression is unaffected by either subtle or gross changes in the uORF peptide. Rather, we find that ribosomes that translate the uORF reinitiate inefficiently and that the intercistronic sequence downstream of the uORF mediates this effect.

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Selected References

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  • Arrick BA, Lee AL, Grendell RL, Derynck R. Inhibition of translation of transforming growth factor-beta 3 mRNA by its 5' untranslated region. Mol Cell Biol. 1991 Sep;11(9):4306–4313. [PMC free article] [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Callis J, Fromm M, Walbot V. Introns increase gene expression in cultured maize cells. Genes Dev. 1987 Dec;1(10):1183–1200. [PubMed]
  • Cao J, Geballe AP. Mutational analysis of the translational signal in the human cytomegalovirus gpUL4 (gp48) transcript leader by retroviral infection. Virology. 1994 Nov 15;205(1):151–160. [PubMed]
  • Cao J, Geballe AP. Translational inhibition by a human cytomegalovirus upstream open reading frame despite inefficient utilization of its AUG codon. J Virol. 1995 Feb;69(2):1030–1036. [PMC free article] [PubMed]
  • Cao J, Geballe AP. Coding sequence-dependent ribosomal arrest at termination of translation. Mol Cell Biol. 1996 Feb;16(2):603–608. [PMC free article] [PubMed]
  • Cao J, Geballe AP. Inhibition of nascent-peptide release at translation termination. Mol Cell Biol. 1996 Dec;16(12):7109–7114. [PMC free article] [PubMed]
  • Damiani RD, Jr, Wessler SR. An upstream open reading frame represses expression of Lc, a member of the R/B family of maize transcriptional activators. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8244–8248. [PMC free article] [PubMed]
  • Degnin CR, Schleiss MR, Cao J, Geballe AP. Translational inhibition mediated by a short upstream open reading frame in the human cytomegalovirus gpUL4 (gp48) transcript. J Virol. 1993 Sep;67(9):5514–5521. [PMC free article] [PubMed]
  • Grant CM, Hinnebusch AG. Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control. Mol Cell Biol. 1994 Jan;14(1):606–618. [PMC free article] [PubMed]
  • Grant CM, Miller PF, Hinnebusch AG. Requirements for intercistronic distance and level of eukaryotic initiation factor 2 activity in reinitiation on GCN4 mRNA vary with the downstream cistron. Mol Cell Biol. 1994 Apr;14(4):2616–2628. [PMC free article] [PubMed]
  • Hill JR, Morris DR. Cell-specific translational regulation of S-adenosylmethionine decarboxylase mRNA. Dependence on translation and coding capacity of the cis-acting upstream open reading frame. J Biol Chem. 1993 Jan 5;268(1):726–731. [PubMed]
  • Hu J, Anderson B, Wessler SR. Isolation and characterization of rice R genes: evidence for distinct evolutionary paths in rice and maize. Genetics. 1996 Mar;142(3):1021–1031. [PMC free article] [PubMed]
  • Janosi L, Shimizu I, Kaji A. Ribosome recycling factor (ribosome releasing factor) is essential for bacterial growth. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4249–4253. [PMC free article] [PubMed]
  • Klein TM, Roth BA, Fromm ME. Regulation of anthocyanin biosynthetic genes introduced into intact maize tissues by microprojectiles. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6681–6685. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. [PMC free article] [PubMed]
  • Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol. 1987 Oct;7(10):3438–3445. [PMC free article] [PubMed]
  • Kozak M. Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems. Mol Cell Biol. 1989 Nov;9(11):5073–5080. [PMC free article] [PubMed]
  • Kozak M. Features in the 5' non-coding sequences of rabbit alpha and beta-globin mRNAs that affect translational efficiency. J Mol Biol. 1994 Jan 7;235(1):95–110. [PubMed]
  • Lloyd AM, Walbot V, Davis RW. Arabidopsis and Nicotiana anthocyanin production activated by maize regulators R and C1. Science. 1992 Dec 11;258(5089):1773–1775. [PubMed]
  • Ludwig SR, Habera LF, Dellaporta SL, Wessler SR. Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7092–7096. [PMC free article] [PubMed]
  • Ludwig SR, Bowen B, Beach L, Wessler SR. A regulatory gene as a novel visible marker for maize transformation. Science. 1990 Jan 26;247(4941):449–450. [PubMed]
  • Luehrsen KR, de Wet JR, Walbot V. Transient expression analysis in plants using firefly luciferase reporter gene. Methods Enzymol. 1992;216:397–414. [PubMed]
  • Luo Z, Sachs MS. Role of an upstream open reading frame in mediating arginine-specific translational control in Neurospora crassa. J Bacteriol. 1996 Apr;178(8):2172–2177. [PMC free article] [PubMed]
  • Luukkonen BG, Tan W, Schwartz S. Efficiency of reinitiation of translation on human immunodeficiency virus type 1 mRNAs is determined by the length of the upstream open reading frame and by intercistronic distance. J Virol. 1995 Jul;69(7):4086–4094. [PMC free article] [PubMed]
  • Merrick WC. Mechanism and regulation of eukaryotic protein synthesis. Microbiol Rev. 1992 Jun;56(2):291–315. [PMC free article] [PubMed]
  • Parola AL, Kobilka BK. The peptide product of a 5' leader cistron in the beta 2 adrenergic receptor mRNA inhibits receptor synthesis. J Biol Chem. 1994 Feb 11;269(6):4497–4505. [PubMed]
  • Peabody DS, Berg P. Termination-reinitiation occurs in the translation of mammalian cell mRNAs. Mol Cell Biol. 1986 Jul;6(7):2695–2703. [PMC free article] [PubMed]
  • Ryoji M, Karpen JW, Kaji A. Further characterization of ribosome releasing factor and evidence that it prevents ribosomes from reading through a termination codon. J Biol Chem. 1981 Jun 10;256(11):5798–5801. [PubMed]
  • Schleiss MR, Degnin CR, Geballe AP. Translational control of human cytomegalovirus gp48 expression. J Virol. 1991 Dec;65(12):6782–6789. [PMC free article] [PubMed]
  • Sleigh MJ. A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells. Anal Biochem. 1986 Jul;156(1):251–256. [PubMed]
  • Song WY, Wang GL, Chen LL, Kim HS, Pi LY, Holsten T, Gardner J, Wang B, Zhai WX, Zhu LH, et al. A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science. 1995 Dec 15;270(5243):1804–1806. [PubMed]
  • Weisshaar B, Armstrong GA, Block A, da Costa e Silva O, Hahlbrock K. Light-inducible and constitutively expressed DNA-binding proteins recognizing a plant promoter element with functional relevance in light responsiveness. EMBO J. 1991 Jul;10(7):1777–1786. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed]

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