Poster abstracts
Poster number 39 submitted by Fiona Fitzgerald
Investigating the role of the DEAD-box helicase Drs1 in assembly of pre-60S subunits in Saccharomyces cerevisiae
Fiona Fitzgerald (Biological Sciences, Carnegie Mellon University), Stephanie Biedka (Biological Sciences, Carnegie Mellon University), Daniel M. Wilson (Biological Sciences, Carnegie Mellon University), Cate McCormick (Biological Sciences, Carnegie Mellon University), John L. Woolford (Biological Sciences, Carnegie Mellon University)
Abstract:
Ribosomes contain a large subunit (LSU) and a small subunit (SSU), made of both ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). rRNA is transcribed in the nucleolus and transits through the nucleus to the cytoplasm, while undergoing folding and binding of r-proteins, enabled by assembly factors (AFs). Properly assembled functional centers within the LSU- the peptidyl transferase center (PTC) and the polypeptide exit tunnel (PET)- are essential for ribosomes to carry out accurate and efficient protein synthesis. Thus, assembly of the PTC and the PET must be closely monitored.
Twelve RNA helicases are necessary for LSU assembly, presumably to chaperone or surveil proper folding of rRNA. RNA helicases utilize binding and hydrolysis of ATP to unwind or remodel RNA or RNP substrates. These helicases contain a core domain, as well as either or both N- and C-terminal extensions (tails). While the catalytic cores of these proteins are more conserved, their tails are less so- presumably providing substrate specificity.
Drs1, a DEAD-box helicase necessary for LSU biogenesis, enters pre-60S ribosomes early, when nascent rRNA is starting to fold, and exits as pre-ribosomes transit from the nucleolus to the nucleoplasm. By learning more about the presence, activity, and specificity of Drs1, we can learn more about early stages of LSU biogenesis, rRNA folding, as well as formation of the PTC and PET.
Using chemical cross-linking, together with truncation mutants, we have found that the C-terminal tail of Drs1 is necessary for its entry into pre-ribosomes, through interactions made with other AFs. We are also able to determine the role that Drs1 plays in pre-ribosome remodeling events through both random and site-directed mutations predicted to alter ATP binding or hydrolysis. In these mutants, Drs1 binds but is not released from pre-ribosomes, preventing pre-mature particles from exiting the nucleolus while also negatively affecting the recycling of various early AFs.
Keywords: helicase, rRNA