RNA helicase activity of the plum pox potyvirus CI protein expressed in Escherichia coli. Mapping of an RNA binding domain.
The plum pox potyvirus (PPV) cylindrical inclusion (CI) protein fused to the maltose binding protein (MBP) has been synthesized in Escherichia coli and purified by affinity chromatography in amylose resin. In the absence of any other viral factors, the fusion product had NTPase, RNA binding and RNA helicase activities. These in vitro activities were not affected by removal of the last 103 amino acids of the CI protein. However, other deletions in the C-terminal part of the protein, although leaving intact all the region conserved in RNA helicases, drastically impaired the ability to unwind dsRNA and to hydrolyze NTPs. A mutant protein lacking the last 225 residues retained the competence to interact with RNA. Further deletions mapped boundaries of the RNA binding domain within residues 350 and 402 of the PPV CI protein. This region includes the arginine-rich motif VI, the most carboxy terminal conserved domain of RNA helicases of the superfamily SF2. These results indicate that NTP hydrolysis is not an essential component for RNA binding of the PPV CI protein.
Fernández
A
A
Centro Nacional de Biotecnología (CSIC), Campus de la Universidad Autónoma de Madrid, Spain.
Laín
S
S
García
J A
JA
D018528:ATP-Binding Cassette Transporters; D017766:Acid Anhydride Hydrolases / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D008320:Maltose; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D043583:Nucleoside-Triphosphatase; D017801:Plum Pox Virus / Q000201:enzymology* / Q000235:genetics; D012313:RNA / Q000378:metabolism*; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D015347:RNA Probes / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D012995:Solubility; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D015347:RNA Probes; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C461599:maltose transport system, E coli; D012313:RNA; D008320:Maltose; D012316:RNA Nucleotidyltransferases; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase; D020365:RNA Helicases