Turning the ribosome into a small-molecule sensor
Arrest Peptides
During translation, nascent proteins pass through a long exit tunnel spanning the large ribosomal subunit before being released into the cytoplasm or delivered to the protein translocation machinery.
Although most proteins can easily complete this journey, some nascent peptides, known as arrest peptides, cause the ribosome that produces them to stall, impacting the expression of downstream genes on the same mRNA. Since stalling sometimes requires specific small molecules, such as amino acids or drugs, arrest peptides are used for metabolite-dependent gene regulation in bacteria and eukaryotes.
A major aim of our group is to understand how arrest peptides program ribosomes to become small-molecule sensors.
Highlights
Small-molecule sensors
We identified a new arrest peptide, called SpeFL, and showed that the amino acid L-ornithine causes ribosomes translating speFL to stall, resulting in the induction of polyamine biosynthesis in γ-proteobacteria (Herrero del Valle et al. (2020) Nat Microbiol). Using cryo-EM, we revealed how the ribosome and nascent SpeFL form a highly selective binding pocket to capture a single L-ornithine molecule, and how this ultimately leads to translational arrest. In collaboration with the Cruz-Vera (Univ. of Alabama in Huntsville), Sachs (Texas A&M) and Mankin and Vázquez-Laslop (Univ. of Illinois at Chicago) groups, we followed this work with the elucidation of the mechanism by which TnaC, an arrest peptide found in Gram-negative bacteria, acts as an L-tryptophan sensor to trigger the production of the signaling molecule indole (van der Stel et al. (2021) Nat Commun). These studies suggest that arrest peptides that detect small molecules with low intrinsic affinity for the ribosome are likely to operate under kinetic control, meaning that high ligand concentrations are required to ensure their efficient capture by the translating ribosome.
L-ornithine recognition by a SpeFL–70S complex. a, Density map of a SpeFL–70S complex. b,c, Density and molecular models of SpeFL and L-ornithine
Deep mutational scan of ErmDL performed by iTP-Seq
Drug-dependent arrest peptides
Arrest peptides can also detect small molecules with high intrinsic affinity for the ribosome, as illustrated by the drug-dependent induction of antibiotic resistance genes in some bacteria. For example, ribosomes translating the leader sequences of erythromycin resistance methyltransferase (erm) genes stall in the presence of macrolide antibiotics. Ribosome stalling induces a rearrangement of the mRNA secondary structure which ultimately leads to the expression of the resistance gene.
In collaboration with the Wilson (Univ. of Hamburg), Mankin and Vázquez-Laslop (Univ. of Illinois at Chicago), and Grubmüller (MPI Göttingen) groups, we showed how macrolide antibiotics are sensed by the arrest peptide ErmDL to induce the expression of the ermD gene (Beckert et al. (2021) Nat Commun). Using inverse toeprinting coupled to next-generation sequencing (iTP-Seq), a profiling method developed in our group, we performed a deep mutational scan of the ErmDL peptide. In combination with structural and biochemical data from our collaborators, our data revealed that ErmDL employs distinct mechanisms to detect and respond to the closely related antibiotics erythromycin (a first-generation macrolide) or telithromycin (a third-generation macrolide known as a ketolide). Revealing the mechanisms by which low doses of drug turn on resistance genes could help design improved antibiotics that fail to induce the expression of these widespread resistance determinants.​
Diversity of metabolite-sensing arrest peptides
Despite recent progress, the full range of metabolites that can be detected by nascent peptides is unknown, and the molecular basis of metabolite sensing by the ribosome is only just beginning to be understood.
Using bioinformatics, cryo-EM and high-throughput tools developed in-house – such as iTP-seq (Seip et al., (2018) Life Sci Alliance) – we continue to systematically address these questions in order to reveal the true extent of metabolite sensing by arrest peptides.