A Highly Conserved Mechanism of Regulated Ribosome Stalling Mediated by Fungal Arginine Attenuator Peptides That Appears Independent of the Charging Status of Arginyl-tRNAs

Abstract

The Arg attenuator peptide (AAP) is an evolutionarily conserved peptide involved in Arg-specific negative translational control. It is encoded as an upstream open reading frame (uORF) in fungal mRNAs specifying the small subunit of Arg-specific carbamoyl phosphate synthetase. We examined the functions of the Saccharomyces cerevisiae CPA1 and Neurospora crassa arg-2 AAPs using translation extracts from S. cerevisiae, N. crassa, and wheat germ. Synthetic RNA containing AAP and firefly luciferase (LUC) sequences were used to program translation; analyses of LUC activity indicated that the AAPs conferred Arg-specific negative regulation in each system. The AAPs functioned either as uORFs or fused in-frame at the N terminus of LUC. Mutant AAPs lacking function in vivo did not function in vitro. Therefore, trans-acting factors conferring AAP-mediated regulation are in both fungal and plant systems. Analyses of ribosome stalling in the fungal extracts by primer extension inhibition (toeprint) assays showed that these AAPs acted similarly to stall ribosomes in the region immediately distal to the AAP coding region in response to Arg. The regulatory effect increased as the Arg concentration increased; all of the arginyl-tRNAs examined appeared maximally charged at low Arg concentrations. Therefore, AAP-mediated Arg-specific regulation appeared independent of the charging status of arginyl-tRNA.

Revealing modularity and organization in the yeast molecular network by integrated analysis of highly heterogeneous genomewide data

“…The integrative power of SAMBA is exemplified by the inclusion of YOR302W in the module, based on the phenotype and the TF binding properties in the module, even though its expression profile is not sufficiently correlated to the module profile. Indeed, YOR302W is CPA1's upstream ORF and is known to function in its translation regulation (16).…”

Roles for transcript leaders in translation and mRNA decay revealed by transcript leader sequencing

“…Thus uAUGs are underrepresented in yeast TLs. Nevertheless, some yeast uAUGs are known to have biological functions as translational regulators of gene expression (e.g., GCN4 [ Mueller and Hinnebusch 1986] and CPA1 [Wang et al 1999]). Consistent with potential regulatory importance, uAUG is the most conserved of all possible uNNN codons in the TL region (Fig.…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…CPA1 control is not mediated by monitoring tRNA Arg charging levels and the Saccharomyces AAP sequence does not contain Arg residues (Wang et al 1999). Hence it is mechanistically distinct from both GCN4 uORF control and the ribosome stalling associated with Trp attenuation in bacteria.…”

“…The presence of high Arg concentrations induces ribosome stalling at the uORF stop codon, with 80S complexes retaining a P-site tRNA-linked nascent peptide within the ribosome exit tunnel. The stalled 80S prevents any PICs that leaky scan through the uORF AUG codon from progressing to the CPA1 AUG codon (Wang et al 1999;Gaba et al 2001). This ensures Cpa1 levels drop when Arg is abundant.…”

“…Many single missense mutations in the uORF eliminate Arg-controlled ribosome stalling (Delbecq et al 2000). Of these, the D13N mutant has been used widely to inform mechanistic understanding (Wang et al 1999;Gaba et al 2001). In contrast to GCN4, most nucleotide sequences around the CPA1 uORF are not important (Delbecq et al 1994).…”