Role of mRNA secondary structure in translational repression of the maize transcriptional activator Lc(1,2).

Lc, a member of the maize (Zea mays) R/B gene family, encodes a basic helix-loop-helix transcriptional activator of the anthocyanin biosynthetic pathway. It was previously shown that translation of the Lc mRNA is repressed by a 38-codon upstream open reading frame (uORF) in the 5' leader. In this study, we report that a potential hairpin structure near the 5'end of the Lc mRNA also represses downstream translation in the rabbit reticulocyte in vitro translation system and in transient transformation assays. Base pairing of the hairpin is important for repression because its destabilization increases translation of the uORF and the downstream ORF. However, translation of the uORF is not required for the hairpin-mediated repression. Instead, the uORF and the 5'-proximal hairpin mediate two independent levels of repression. Although the uORF represses downstream translation due to inefficient reinitiation of ribosomes that translate uORF, the hairpin inhibits ribosome loading at the 5' end of the mRNA.

[1]  M. Fromm,et al.  Regulation of anthocyanin biosynthetic genes introduced into intact maize tissues by microprojectiles. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[2]  V. Walbot,et al.  Transient expression analysis in plants using firefly luciferase reporter gene. , 1992, Methods in enzymology.

[3]  S. Wessler,et al.  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. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[4]  C. Tonelli,et al.  Molecular homology among members of the R gene family in maize. , 1993, The Plant journal : for cell and molecular biology.

[5]  V. Walbot,et al.  Introns increase gene expression in cultured maize cells. , 1987, Genes & development.

[6]  M. Kozak.  Features in the 5′ non-coding sequences of rabbit α and β-globin mRNAs that affect translational efficiency , 1994 .

[7]  M. Kozak.  Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[8]  F Sherman,et al.  mRNA structures influencing translation in the yeast Saccharomyces cerevisiae , 1988, Molecular and cellular biology.

[9]  M. Kozak.  Primer extension analysis of eukaryotic ribosome-mRNA complexes. , 1998, Nucleic acids research.

[10]  M. Kozak.  Initiation of translation in prokaryotes and eukaryotes. , 1999, Gene.

[11]  S. Wessler,et al.  Inefficient Reinitiation Is Responsible for Upstream Open Reading Frame–Mediated Translational Repression of the Maize R Gene , 1998, Plant Cell.

[12]  E. Conway de Macario,et al.  Identification of a grpE heat-shock gene homolog in the archaeon Methanosarcina mazei. , 1994, Journal of molecular biology.

[13]  A. Hinnebusch,et al.  Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control , 1994, Molecular and cellular biology.

[14]  D. Crothers,et al.  Improved estimation of secondary structure in ribonucleic acids. , 1973, Nature: New biology.

[15]  A. Fellous,et al.  Co‐assembly properties of higher plant microtubule‐associated proteins with purified brain and plant tubulins , 1993 .

[16]  M. Sleigh,et al.  A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells. , 1986, Analytical biochemistry.

[17]  S. Wessler,et al.  A Regulatory Gene as a Novel Visible Marker for Maize Transformation , 1990, Science.

[18]  A. Geballe,et al.  Translational Control by an Upstream Open Reading Frame in the HER-2/neu Transcript* , 1999, The Journal of Biological Chemistry.

[19]  M. Kozak,et al.  Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs , 1989, Molecular and cellular biology.

[20]  S. Goff,et al.  Transactivation of anthocyanin biosynthetic genes following transfer of B regulatory genes into maize tissues. , 1990, The EMBO journal.

[21]  M. Kozak,et al.  Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[22]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[23]  S. Wessler,et al.  An upstream open reading frame represses expression of Lc, a member of the R/B family of maize transcriptional activators. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[24]  G. Sarkar,et al.  The "megaprimer" method of site-directed mutagenesis. , 1990, BioTechniques.