Dept. of Environmental and Biomolecular Systems
Oregon Health & Science University
Neurospora crassa arg-2 gene encodes the small subunit of arginine specific carbamoyl phosphate synthethase, the first enzyme in fungal arginine (Arg) biosynthesis. The arg-2 mRNA contains an upstream open reading frame (uORF) specifying an evolutionarily conserved 24-residue peptide called the arginine attenuator peptide (AAP). Synthesis of the AAP causes ribosomes to stall on the mRNA in the presence of high concentrations of Arg. The amino acid sequence of the AAP, and not the sequence of its coding region, is responsible for this regulation. Scanning mutagenesis within the evolutionarily conserved region revealed that some residues are more important than others for the AAP to function. While most known nascent peptides that regulate translation are found encoded as uORFs or as N-terminal leader peptides, the AAP can exert regulatory function whether placed near the N-terminus or internally within a large polypeptide. The AAPâs peptide sensing features are conserved due to regulated stalling of fungal, plant, and animal ribosomes in response to Arg. That the AAP functions as an internal domain to regulate elongation in response to Arg establishes that such domains can provide a means of controlling translational elongation. The minimal sequences required for AAP to function as an internal domain was revealed by systematic deletion of its natural N- and C-terminal regions. Comparative analysis of the AAP with other fungi showed that the evolutionarily conserved region of the peptide is required for regulation. This minimal domain functions when placed as a uORF as seen by toeprint assay. Analyses of Arg analogs provided key insights in the structural requirements for Argâs role in regulation. These results taken together provide a detailed picture of the requirements for Arg-specific regulation mediated by the AAP.
OGI School of Science and Engineering
Spevak, Christina C., "Specific requirements for translational regulation by a nascent peptide that stalls ribosomes in response to arginine" (2006). Scholar Archive. 42.