Poster abstracts
Poster number 81 submitted by Amber LaPeruta
Construction and inspection of the polypeptide exit tunnel in the yeast 60S ribosomal subunit
Amber J. LaPeruta (Carnegie Mellon University), Dan M. Wilson (Carnegie Mellon University), John L. Woolford (Carnegie Mellon University)
Abstract:
Assembly of 60S ribosomal subunits in eukaryotes proceeds in a hierarchical fashion involving the entry and exit of approximately 80 assembly factors. Generation of mature 60S particles requires the timely construction of multiple functional centers, which may require passing quality control checkpoints. Among many of these functional centers is the polypeptide exit tunnel (PET). Recent cryo-EM structures of pre-ribosomal particles purified using Nog2-TAP, Arx1-TAP and Nmd3-TAP have given us new insights into the mechanisms involved in the assembly of this complex and interactive conduit which, until now, have remained elusive. In all three of these structures a theme arises: the C-terminal extensions of three assembly factors (first Nog1 followed by Rei1 then Reh1) probe the entire length of the exit tunnel, reaching to the peptidyl transferase center (PTC). These tails appear to interact with the rRNA lining of the PET and the tentacle-like extensions of ribosomal proteins L17 (μL22) and L4 (μL4), which form a constriction site in the PET. The mutually exclusive occupation of the PET by various assembly factors from Nog2’s lifetime onward raises several hypotheses. The C-terminal extensions of Nog1, Rei1 and Reh1 may function as scaffolds for the folding of rRNA in and around the exit tunnel. Alternatively, they may act as tunnel inspectors or prevent premature function of incomplete ribosomes. To gain insight into the function of these extensions and the assembly of the PET as a whole, we are manipulating the C-terminal extensions of Nog1, Rei1 and Reh1 as well as the β-hairpin of L17 and the extended loop of L4 in S. cerevisiae using deletions, substitutions, and the addition of bulky moieties. These mutant strains are being assessed for growth, polysome profile, and rRNA processing defects as well as by purification and analysis of pre-ribosomal composition.
Keywords: Ribosome, Polypeptide Exit Tunnel, Assembly