Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

Periodic Report Summary 1 - TRANSARREST (Translational Regulation of Gene Expression by the Nascent Polypeptide Chain)

During protein synthesis, the elongating polypeptide chain travels through the nascent polypeptide exit tunnel, a long cavity spanning the large subunit of the ribosome. Although this was previously deemed a passive process, it is now established that certain peptide sequences interact with the ribosomal RNA (rRNA) and proteins lining the walls of the exit tunnel to pause or arrest protein synthesis. In many cases, translational arrest is dependent on a low molecular weight ligand – such as an amino acid or an antibiotic – that is sensed by ribosomes engaged in the synthesis of a specific arrest peptide.

In order to gain a deeper insight into this process, I have chosen to focus on two complementary objectives:

Objective 1. Understand the mechanisms through which nascent chains regulate the ribosome by obtaining high-resolution X-ray or cryo-EM structures of stalled ribosome nascent chain complexes (RNCs). Current strategies for preparing RNCs rely on cell-free expression systems and produce samples of varying quality and stoichiometry. Consequently, I sought to prepare peptidyl-tRNAs directly by attaching chemically synthesized peptides onto tRNAs expressed in vivo, using small RNA enzymes known as flexizymes. Peptidyl-tRNAs obtained in this manner may be mixed with the ribosome to form RNCs, thus sidestepping some of the problems stemming from ribosomal peptide synthesis. Work on this objective has so far focused on two distinct biological systems: (i) translational arrest in the presence of macrolide antibiotics by peptides carrying a short (R/K)x(R/K) arrest motif, and (ii) polyproline-dependent translational arrest and stalled ribosome rescue by elongation factor EF-P.

In addition, our crystallographic analyses of peptide-mediated translational inhibition took us into an unexpected direction, as we demonstrated that certain proline-rich antimicrobial peptides (PrAMPs) produced by the host immune response of insects and mammals block translation by binding to the exit tunnel of the bacterial ribosome (Seefeldt et al. (2016) Nucleic Acids Res 44, 2429; Seefeldt et al. (2015) Nat Struct Mol Biol 22, 470). The overlapping binding sites for arrest peptides, PrAMPs and antibiotics that target the large ribosomal subunit suggest that an increased understanding of these three types of ribosomal ligands could help us design improved translation inhibitors.

Objective 2. Explore the functional diversity of nascent chain-mediated phenomena by developing an in vitro method to identify nascent peptides that arrest translation with the help of a small molecular weight ligand. The method developed in my group, which is referred to here as inverse toeprinting, directly links the translational arrest phenotype to the genotype that causes it, enabling us to simultaneously monitor the stalling potential of >1011 peptides in a high-throughput fashion. Using this approach, we have begun to re-evaluate the primary sequence determinants underlying drug-dependent translational arrest by the Erm leader peptides. These comprise several families of unrelated arrest peptides involved in the acquisition of resistance against macrolide-lincosamide-streptogramin-B antibiotics.

By integrating the structure and sequence data obtained from these studies, we seek to decipher the rules underlying peptide-mediated translational arrest and to systematically assess its biological extent and distribution in nature.

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