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Project Progress

Ribosome peptide exit tunnel plays a crucial role in the functioning of ribosomes across all domains of life. ^{1 2 3} Before the transition of nascent peptides to mature functional proteins, they must travel through the functionally conserved peptide exit tunnel. ⁴ Additionally, the latent chaperone activity of the exit tunnel ^{5 6} suggests its role in ribosomal evolution, in the transition from short non-structured peptides to extant globular proteins. The wall of the tunnel is constructed mostly from RNA. As high as 80% of the tunnel is RNA in some species. ⁴ Our objective is to gain an understanding of the molecular basis of the latent chaperone activity and the preferential construction of the ribosome exit tunnel from the RNA component of the ribosome. Toward this end we have designed ketolide-peptide compounds (peptolides) to probe the mechanisms employed by the ribosome to, (i) facilitate in-tunnel folding of nascent peptides and (ii) distinguish between some peptide sequences while facilitating unhindered passage of the vast majority of peptides through the peptide exit tunnel.

Research Accomplishments:

We have synthesized seven peptolides as probes to explore the passage of nascent peptide through the ribosome exit tunnel. Using a translation inhibition assay, we have established that some of these peptolides specifically differentiate between the prokaryote and eukaryote ribosomes, while others have less specificity. The disparity in the ribosome recognition is mainly due to the peptide component of the peptolides. In collaboration with Prof. Christine Dunham, we have obtained co-crystals of two of these peptolides on E. coli 70S. A partial refinement of the structure of one of the peptolides (DB1106) revealed that DB1106 binds within the exit tunnel where its five-member ring makes an intriguing stacking interaction with His69 of L4

Our near term goal is to continue our structure refinements to elucidate the binding interactions of the peptolide peptide moieties within the exit tunnel. A facile footprinting assay will be developed to map the paths of these and the new generation of probes bearing alternative head group. Also, a manuscript describing our current observation is in preparation.

References:
1. Nakatogawa, H.; Ito, K., The ribosomal exit tunnel functions as a discriminating gate. Cell 2002, 108 (5), 629-36.
2. Bashan, A.; Yonath, A., Ribosome crystallography: catalysis and evolution of peptide-bond formation, nascent chain elongation and its co-translational folding. Biochem Soc Trans 2005, 33 (Pt 3), 488-92.
3. Seidelt, B.; Innis, C. A.; Wilson, D. N.; Gartmann, M.; Armache, J. P.; Villa, E.; Trabuco, L. G.; Becker, T.; Mielke, T.; Schulten, K.; Steitz, T. A.; Beckmann, R., Structural insight into nascent polypeptide chain-mediated translational stalling. Science 2009, 326 (5958), 1412-5.
4. Nissen, P.; Hansen, J.; Ban, N.; Moore, P. B.; Steitz, T. A., The structural basis of ribosome activity in peptide bond synthesis. Science 2000, 289 (5481), 920-30.
5. Kosolapov, A.; Deutsch, C., Tertiary interactions within the ribosomal exit tunnel. Nat Struct Mol Biol 2009, 16 (4), 405-11.
6. Tu, L. W.; Deutsch, C., A folding zone in the ribosomal exit tunnel for Kv1.3 helix formation. J Mol Biol 396 (5), 1346-60.