Abstract
The protein encoded by the Opaque-2 (O2) gene is a transcription factor, translated from an mRNA that possesses an unusually long 5' leader sequence containing three upstream open reading frames (uORFs). The efficiency of translation of O2 mRNA has been tested in vivo by a transient assay in which the level of activation of the b32 promoter, a natural target of O2 protein, is measured. We show that uORF-less O2 alleles possess a higher transactivation value than the wild-type allele and that the reduction in transactivation due to the uORFs is a cis-dominant effect. The data presented indicate that both uORF1 and uORF2 are involved in the reducing effect and suggest that both are likely to be translated.
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- Di Fonzo N., Hartings H., Brembilla M., Motto M., Soave C., Navarro E., Palau J., Rhode W., Salamini F. The b-32 protein from maize endosperm, an albumin regulated by the O2 locus: nucleic acid (cDNA) and amino acid sequences. Mol Gen Genet. 1988 Jun;212(3):481–487. doi: 10.1007/BF00330853. [DOI] [PubMed] [Google Scholar]
- Elgin S. C. Methods for isolation and characterization of nonhistone chromosomal proteins. Methods Enzymol. 1975;40:144–160. doi: 10.1016/s0076-6879(75)40013-1. [DOI] [PubMed] [Google Scholar]
- Entwistle J., Knudsen S., Müller M., Cameron-Mills V. Amber codon suppression: the in vivo and in vitro analysis of two C-hordein genes from barley. Plant Mol Biol. 1991 Dec;17(6):1217–1231. doi: 10.1007/BF00028737. [DOI] [PubMed] [Google Scholar]
- Entwistle J. Primary structure of a C-hordein gene from barley. Carlsberg Res Commun. 1988;53(4):247–258. doi: 10.1007/BF02907181. [DOI] [PubMed] [Google Scholar]
- Hatfield D. L., Smith D. W., Lee B. J., Worland P. J., Oroszlan S. Structure and function of suppressor tRNAs in higher eukaryotes. Crit Rev Biochem Mol Biol. 1990;25(2):71–96. doi: 10.3109/10409239009090606. [DOI] [PubMed] [Google Scholar]
- Hinnebusch A. G. Involvement of an initiation factor and protein phosphorylation in translational control of GCN4 mRNA. Trends Biochem Sci. 1990 Apr;15(4):148–152. doi: 10.1016/0968-0004(90)90215-w. [DOI] [PubMed] [Google Scholar]
- Hinnebusch A. G. Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol Rev. 1988 Jun;52(2):248–273. doi: 10.1128/mr.52.2.248-273.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Kozak M. How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell. 1978 Dec;15(4):1109–1123. doi: 10.1016/0092-8674(78)90039-9. [DOI] [PubMed] [Google Scholar]
- Kozak M. Selection of initiation sites by eucaryotic ribosomes: effect of inserting AUG triplets upstream from the coding sequence for preproinsulin. Nucleic Acids Res. 1984 May 11;12(9):3873–3893. doi: 10.1093/nar/12.9.3873. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Liu C. C., Simonsen C. C., Levinson A. D. Initiation of translation at internal AUG codons in mammalian cells. Nature. 1984 May 3;309(5963):82–85. doi: 10.1038/309082a0. [DOI] [PubMed] [Google Scholar]
- Lohmer S., Maddaloni M., Motto M., Di Fonzo N., Hartings H., Salamini F., Thompson R. D. The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene. EMBO J. 1991 Mar;10(3):617–624. doi: 10.1002/j.1460-2075.1991.tb07989.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lütcke H. A., Chow K. C., Mickel F. S., Moss K. A., Kern H. F., Scheele G. A. Selection of AUG initiation codons differs in plants and animals. EMBO J. 1987 Jan;6(1):43–48. doi: 10.1002/j.1460-2075.1987.tb04716.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marth J. D., Overell R. W., Meier K. E., Krebs E. G., Perlmutter R. M. Translational activation of the lck proto-oncogene. Nature. 1988 Mar 10;332(6160):171–173. doi: 10.1038/332171a0. [DOI] [PubMed] [Google Scholar]
- 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]
- Sayers J. R., Schmidt W., Eckstein F. 5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1988 Feb 11;16(3):791–802. doi: 10.1093/nar/16.3.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schmidt R. J., Burr F. A., Aukerman M. J., Burr B. Maize regulatory gene opaque-2 encodes a protein with a "leucine-zipper" motif that binds to zein DNA. Proc Natl Acad Sci U S A. 1990 Jan;87(1):46–50. doi: 10.1073/pnas.87.1.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schmidt R. J., Burr F. A., Burr B. Transposon tagging and molecular analysis of the maize regulatory locus opaque-2. Science. 1987 Nov 13;238(4829):960–963. doi: 10.1126/science.2823388. [DOI] [PubMed] [Google Scholar]
- Sedman S. A., Good P. J., Mertz J. E. Leader-encoded open reading frames modulate both the absolute and relative rates of synthesis of the virion proteins of simian virus 40. J Virol. 1989 Sep;63(9):3884–3893. doi: 10.1128/jvi.63.9.3884-3893.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Singh K., Tokuhisa J. G., Dennis E. S., Peacock W. J. Saturation mutagenesis of the octopine synthase enhancer: correlation of mutant phenotypes with binding of a nuclear protein factor. Proc Natl Acad Sci U S A. 1989 May;86(10):3733–3737. doi: 10.1073/pnas.86.10.3733. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Soave C., Tardani L., Di Fonzo N., Salamini F. Zein level in maize endosperm depends on a protein under control of the opaque-2 and opaque-6 loci. Cell. 1981 Dec;27(2 Pt 1):403–410. doi: 10.1016/0092-8674(81)90423-2. [DOI] [PubMed] [Google Scholar]
- Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
- Thach R. E. Cap recap: the involvement of eIF-4F in regulating gene expression. Cell. 1992 Jan 24;68(2):177–180. doi: 10.1016/0092-8674(92)90461-k. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Töpfer R., Matzeit V., Gronenborn B., Schell J., Steinbiss H. H. A set of plant expression vectors for transcriptional and translational fusions. Nucleic Acids Res. 1987 Jul 24;15(14):5890–5890. doi: 10.1093/nar/15.14.5890. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Viotti A., Balducci C., Weil J. H. Adaptation of the tRNA population of maize endosperm for zein synthesis. Biochim Biophys Acta. 1978 Jan 26;517(1):125–132. doi: 10.1016/0005-2787(78)90040-0. [DOI] [PubMed] [Google Scholar]
- Wandelt C., Feix G. Sequence of a 21 kd zein gene from maize containing an in-frame stop codon. Nucleic Acids Res. 1989 Mar 25;17(6):2354–2354. doi: 10.1093/nar/17.6.2354. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]