Investigating the N‐ and C‐terminal Extensions of the Yeast DEAD‐box Protein Dbp6

Abstract

DEAD‐box proteins are RNA‐dependent ATPases that are involved in all aspects of RNA metabolism. Also known as DEAD‐box helicases, these enzymes bind RNA and ATP and unwind short RNA duplexes via a highly conserved helicase core. N‐ and C‐terminal extensions that flank this core differ in both length and composition between DEAD‐box proteins and have been shown to contribute to RNA specificity and binding, thereby conferring the diverse cellular roles of DEAD‐box proteins. Here, we are investigating the 192 amino acid N‐terminal and 63 amino acid C‐terminal extensions of Dbp6, a yeast DEAD‐box protein that is required for 60S ribosome biogenesis. To find the minimum functional length of the flanking regions, we have systematically designed truncation constructs and observed their effects on the growth of yeast cells in which endogenous Dbp6 has been depleted. Our results indicate that deletion of up to 168 amino acids at the N‐terminus (ΔN168) or 27 amino acids at the C‐terminus (ΔC27) give either wild‐type level or slow growth. However, removal of five more residues at the N‐terminus or two more at the C‐terminus results in a lethal phenotype. Interestingly, even though Dbp6 ΔN143 and Dbp6 ΔC21 each have near normal growth, the double truncation construct Dbp6 ΔN143ΔC21 has no growth. We are currently conducting Western blot analysis to confirm that Dbp6 is indeed being expressed in these yeast cells with growth phenotypes. Follow‐up experiments include measuring the ATP and/or RNA binding and RNA duplex unwinding activities of Dbp6 truncation constructs in vitro. Since most of the yeast DEAD‐box proteins have orthologs in humans and many of those proteins have been implicated in cancer development or progression, we hope that these insights into the molecular workings of Dbp6 can contribute to our broader understanding of DEAD‐box proteins and their uses as potential cancer therapeutic targets.Support or Funding InformationEckerd College Natural Sciences Summer Research Program (NSSRP)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.