Genetic and biochemical analysis of a conserved DEAD-box helicase motif

DEAD-box proteins are non-processive helicases that use ATP to power unwinding of short RNA duplexes in essential steps of RNA metabolism. Despite acting on a diverse range of RNA substrates, DEAD-box proteins share a helicase core defined by 13 highly conserved amino acid motifs. Here we use S. cerevisiae Mss116p, a mitochondrial helicase required for efficient splicing of group I and group II introns, to probe the role of the conserved motif Va. We combine saturation mutagenesis, in vivo selection, and next-generation sequencing as part of a novel, high-throughput approach to identify functional mutants of motif Va and individual amino acids that are crucial for Mss116p function. We use in vitro assays to further dissect the effects of mutating a highly-conserved motif Va residue, showing that motif Va plays an important role in controlling ATP affinity and coupling of RNA and ATP binding. Our results provide novel insights into the role of a poorly understood DEAD-box motif and establish a useful tool for in-depth analysis of conserved protein motifs

Author dc:creator

• Gilman, Benjamin Douglas

Contributors dc:contributor

• Russell, Rick, 1969-
• Lambowitz, Alan
• Browning, Karen
• Stevens, Scott
• Davies, Bryan

• RNA helicase
• DEAD-box
• Mss116
• Mss116p
• Motif Va

Language dc:language

en

Identifier

http://dx.doi.org/10.26153/tsw/42594

OAI identifier oai:identifier

oai:tdl-ir.tdl.org:2152/115696