Research Interests:
We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe... more
We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be...
Research Interests:
RNA helicases are essential for virtually all cellular processes, however, their regulation is poorly understood. The activities of eight RNA helicases are required for pre-mRNA splicing. Amongst these, Brr2p is unusual in having two... more
RNA helicases are essential for virtually all cellular processes, however, their regulation is poorly understood. The activities of eight RNA helicases are required for pre-mRNA splicing. Amongst these, Brr2p is unusual in having two helicase modules, of which only the amino-terminal helicase domain appears to be catalytically active. Using genetic and biochemical approaches, we investigated interaction of the carboxy-terminal helicase module, in particular the carboxy-terminal Sec63-2 domain, with the splicing RNA helicase Prp16p. Combining mutations in BRR2 and PRP16 suppresses or enhances physical interaction and growth defects in an allele-specific manner, signifying functional interactions. Notably, we show that Brr2p Sec63-2 domain can modulate the ATPase activity of Prp16p in vitro by interfering with its ability to bind RNA. We therefore propose that the carboxy-terminal helicase module of Brr2p acquired a regulatory function that allows Brr2p to modulate the ATPase activity...