The newly discovered Q motif of DEAD-box RNA helicases regulates RNA-binding and helicase activity.
DEAD-box proteins are the most common RNA helicases, and they are associated with virtually all processes involving RNA. They have nine conserved motifs that are required for ATP and RNA binding, and for linking phosphoanhydride cleavage of ATP with helicase activity. The Q motif is the most recently identified conserved element, and it occurs approximately 17 amino acids upstream of motif I. There is a highly conserved, but isolated, aromatic group approximately 17 amino acids upstream of the Q motif. These two elements are involved in adenine recognition and in ATPase activity of DEAD-box proteins. We made extensive analyses of the Q motif and upstream aromatic residue in the yeast translation-initiation factor Ded1. We made site-specific mutations and tested them for viability in yeast. Moreover, we purified various mutant proteins and obtained the Michaelis-Menten parameters for the ATPase activities. We also measured RNA affinities and strand-displacement activities. We find that the Q motif not only regulates ATP binding and hydrolysis but also regulates the affinity of the protein for RNA substrates and ultimately the helicase activity.
Cordin
Olivier
O
Département Microbiologie et Médecine Moléculaire, Centre Médical Universitaire, Geneva, Switzerland.
Tanner
N Kyle
NK
Doère
Monique
M
Linder
Patrick
P
Banroques
Josette
J
D000225:Adenine / Q000737:chemistry / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic / Q000737:chemistry; D016384:Consensus Sequence; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D039601:Eukaryotic Initiation Factor-4A / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D020365:RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012331:RNA, Fungal / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D024322:Amino Acids, Aromatic; D005656:Fungal Proteins; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D039601:Eukaryotic Initiation Factor-4A; D000251:Adenosine Triphosphatases; D020365:RNA Helicases; D007930:Leucine; D000225:Adenine; D000409:Alanine; D005998:Glycine