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

Abstract

DEAD-box proteins are RNA-dependent ATPases that are involved in all aspects of RNA metabolism. Even though these enzymes are highly conserved in both sequence and structure, each DEAD-box protein has a unique intracellular function and is therefore essential. To gain insight into this enzymatic specificity, we are investigating the significance of the 192 amino acid N-terminal and 63 amino acid C-terminal extensions of Dbp6, a protein required for yeast ribosome assembly. To find the minimum functional length of the flanking region, serial truncation constructs were designed and their effect on the growth of yeast cells in which endogenous Dbp6 has been depleted was observed. The 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. Western blot analysis supported the hypothesis that a non-functional protein was present. We are currently cloning the truncation constructs into bacterial expression vectors in order to over-express, purify, and test the enzymatic activity of the truncated proteins. Additionally, we are using site-directed mutagenesis to determine the effect of single amino acid mutations in the N-terminus and conserved core. Future experiments include measuring the ATP and RNA binding and RNA duplex unwinding activities of truncated Dbp6 in vitro. Understanding the enzymatic function of Dbp6 in vitro will elucidate its specific cellular roles and allow for further research on the involvement of Dbp6 and its human ortholog DDX51 in cell proliferation, viability, and cancer signaling. Gaining insight into the normal and impaired functions of DDX51 through this yeast model system will hopefully lead to future research on the potential therapeutic targeting of DEAD-box proteins in cancer treatment.