Sequence Requirements for Ribosome Stalling by the Arginine Attenuator Peptide

Abstract

The 5 regions of eukaryotic mRNAs often contain upstream open reading frames (uORFs). The Neurospora crassa arg-2 uORF encodes the 24-residue arginine attenuator peptide (AAP). This regulatory uORF-encoded peptide, which is evolutionarily conserved in fungal transcripts specifying an arginine biosynthetic enzyme, functions as a nascent peptide within the ribosomal tunnel and negatively regulates gene expression. The nascent AAP causes ribosomes to stall at the uORF stop codon in response to arginine, thus, blocking ribosomes from reaching the ARG-2 initiation codon. Here scanning mutagenesis with alanine and proline was performed to systematically determine which AAP residues were important for conferring regulation. Changing many of the most highly conserved residues (Asp-12, Tyr-13, Lys-14, and Trp-19) abolished regulatory function. The minimal functional domain of the AAP was determined by positioning AAP sequences internally within a large polypeptide. Pulse-chase analyses revealed that residues 9 -20 of the AAP composed the minimal domain that was sufficient to confer regulatory function. An extensive analysis of predicted fungal AAPs revealed that the minimal functional domain of the N. crassa AAP corresponded closely to the region that was most highly conserved among the fungi. We also observed that the tripeptide RGD could function similarly to arginine in triggering AAP-mediated ribosome stalling. These studies provide a better understanding of the elements required for a nascent peptide and a small regulatory molecule to control translational processes.

The Arginine Attenuator Peptide Interferes with the Ribosome Peptidyl Transferase Center

“…most efficient at achieving and/or maintaining this proper register. Consistent with this idea, of 120 uORF-encoded AAPs identified by evolutionary conservation, the C-terminal regions of 119 are at least as long as the N. crassa AAP C terminus, and none is more than two residues longer (49).…”

“…ErmCL, ErmAL1, and SecM each also require nearby amino acids (Ϫ2 relative to the nascent peptide C terminus) for stalling to occur (44,63). In contrast, AAP residues 9 to 20 are sufficient to confer regulatory function; moreover, AAP residues 21 to 24 can all be replaced with Ala, and AAP function is retained (49). Furthermore, the evolutionary conservation of residues of the AAP beyond Arg-20 is relatively low.…”

“…Arg-regulated stalling by the AAP requires that Arg has a free N terminus (49). An Arg-Gly-Asp (RGD) tripeptide, which can induce AAP-mediated ribosome stalling (49) and induce a change in the AAP's relative conformation in the ribosome (61), also was inhibitory to puromycin release, albeit more weakly than Arg (Fig.…”

Arginine Changes the Conformation of the Arginine Attenuator Peptide Relative to the Ribosome Tunnel

“…The mRNAs that contained selectively placed amber stop codons in the AAP coding region were truncated after a C-terminal AAP sense-codon to generate stable ribosome-nascent chain complexes (RNCs) in wheat germ and N. crassa cell-free translation systems. In the absence of other constraints on translation, the final codon of the truncated RNA will be in the ribosomal P site and the amino acid corresponding to this codon will be placed in the nascent chain 17; 32 . To visualize these nascent peptides, additional Met-codons were added at the N-terminus of the AAP (Fig.…”

“…1) are absolutely conserved in all AAPs identified so far 16; 17 and mutations at either position eliminate stalling 17 . A difference between the AAP and these bacterial NSPs, however, is that there is no demonstrated requirement for the AAP to have a specific residue at the PTC when stalling occurs 4; 15; 17; 18; 19; 20 . Despite conservation of length, there is considerable sequence divergence of AAPs at their C-termini 16; 17 .…”