| Maize R genes encode a small family of basic helix-loop-helix transcriptional activators of the anthocyanin biosynthetic pathway. The 235-nucleotide leader of the R gene Lc contains a 38-codon upstream open reading frame (uORF) and a potential hairpin structure (Δ G = –15.6 kcal/mol) near the 5′ end. The work presented in this dissertation demonstrates that translation of the Lc mRNA is repressed by the presence of the uORF and the 5′ hairpin.; The uORF is shown to repress downstream translation both in the rabbit reticulocyte in vitro 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. It has been demonstrated that translation of the uORF is required for repression, and that increasing translation of the uORF peptide decreases downstream gene expression. However, repression is unaffected by either minor or major changes in the uORF peptide. Rather, the data presented indicate that repression is due to inefficient reinitiation of ribosomes that translate the uORF, and that the intercistronic sequence downstream of the uORF mediates inefficient reinitiation.; Secondary structure provides another mechanism of translational control of Lc. The potential hairpin structure near the 5 ′end of the Lc mRNA is found to repress downstream translation both in vitro and in vivo. The base pairing of the hairpin is important for repression, and mutations that disrupt the base pairing increase downstream gene expression whether or not the uORF is also present. For dicistronic mRNAs, destabilization of the hairpin structure increases translation of both the uORF and the downstream ORF. Monocistronic RNA containing a mutated hairpin region is translated by more ribosomes than its wild-type counterpart.; Taken together, our data lead us to propose a model in which translation of the Lc mRNA is repressed by two independent mechanisms. While the uORF represses downstream translation due to inefficient reinitiation of ribosomes that translate uORF, the hairpin inhibits translation by reducing ribosome loading at the 5′ end of the mRNA. |
