Shedding Light on the DEAD: Preliminary Investigation into Dbp6, a DEAD-box Protein Required for Ribosome Assembly
Ribosomes, the essential RNA-protein machines responsible for catalyzing protein synthesis are made up of two subunits: the large 60S subunit and the small 40S subunit. In Saccharomyces cerevisiae, more commonly known as Baker's yeast, the 60S subunit contains three ribosomal RNAs (rRNAs; 5S, 5.8S and 25S) while the 40S contains one rRNA (18S). Along with the four rRNAs, yeast ribosomes contain 78 ribosomal proteins. To ensure that the ribosome components are assembled correctly each and every time, a macromolecular machinery of ~200 accessory factors is required during ribosome biogenesis. These different factors include RNA-binding proteins, protein kinases, GTPases, ATPases, endonucleases and RNA helicases. DEAD-box proteins, named for a highly conserved D-E-A-D motif in their primary sequence, are the largest class of RNA helicases. DEAD-box protein 6 (Dbp6) is a DEAD-box protein involved in ribosome biogenesis in S. cerevisiae. In vivo depletion of Dbp6 results in a decrease in 60S subunits due to the depletion of the 27S and 7S rRNA precursors to the 25S and 5.8S rRNAs, respectively. Localized in the nucleus, Dbp6 is known to form a complex with two additional assembly factors: ribosome assembly factor 3 (Rsa3) and nucleolar protein 8 (Nop8). In order to achieve high specificity in vivo, it is postulated that DEAD-box proteins interact with co-factors; Rsa3 and Nop8 may be Dbp6 specificity factors. In order to further investigate Dbp6, a sequence analysis was performed, first comparing Dbp6 to DEAD-box proteins in other forms of life followed by a comparison to other DEAD-box proteins in yeast. Dbp6 is highly conserved in eukaryotes but not in bacteria. It also contains nine out of the ten highly conserved DEAD-box protein motifs and has a notably short C-terminus in comparison to its yeast homologs. These sequence alignments can be used in the future to design truncations and mutations for a functional analysis. To begin analyzing Dbp6 in yeast, wild-type Dbp6 was cloned into the yeast expression vector pRS416GPD. Bacterial colony PCR revealed that the wild-type Dbp6 insert was not successfully ligated into the expression vector. Trouble-shooting attempts have indicated that the restriction digest and subsequent ligation is not effective. During the molecular biology of the project, a preliminary investigation into co-factors of Dbp6 was initiated. To being, expression conditions for Nop8 were optimized. The comparison of two different cell lines, BL21 and Rosetta, and varying concentrations of isopropyl β-D-1-thiogalactopyranoside (IPTG), 0.2 mM and 1 mM, indicated that inducing Rosetta cells with 0.2 mM IPTG overnight at 18ËšC is the best condition for maximum Nop8 expression. Future work involves overexpressing and purifying a large scale preparation of Nop8, cloning and overexpressing Rsa3 and the eventual in vitro testing of binding interactions between Nop8, Rsa3 and Dbp6.
© Copyright 2013 Kari Martyniak