Vaccinia virus RNA helicase: nucleic acid specificity in duplex unwinding.
Vaccinia virus RNA helicase (NPH-II) catalyzes nucleoside triphosphate-dependent unwinding of duplex RNAs containing a single-stranded 3' RNA tail. In this study, we examine the structural features of the nucleic acid substrate that are important for helicase activity. Strand displacement was affected by the length of the 3' tail. Whereas NPH-II efficiently unwound double-stranded RNA substrates with 19- or 11-nucleotide (nt) 3' tails, shortening the 3' tail to 4 nt reduced unwinding by an order of magnitude. Processivity of the helicase was inferred from its ability to unwind a tailed RNA substrate containing a 96-bp duplex region. NPH-II exhibited profound asymmetry in displacing hybrid duplexes composed of DNA and RNA strands. A 34-bp RNA-DNA hybrid with a 19-nt 3' RNA tail was unwound catalytically, whereas a 34-bp DNA-RNA hybrid containing a 19-nt 3' DNA tail was 2 orders of magnitude less effective as a helicase substrate. NPH-II was incapable of displacing a 34-bp double-stranded DNA substrate of identical sequence. 3'-Tailed DNA molecules with 24- or 19-bp duplex regions were also inert as helicase substrates. On the basis of current models for RNA-DNA hybrid structures, we suggest the following explanation for these findings. (i) Unwinding of duplex nucleic acids by NPH-II is optimal when the polynucleotide strand of the duplex along which the enzyme translocates has adopted an A-form secondary structure, and (ii) a B-form secondary structure impedes protein translocation through DNA duplexes.
Gross
C H
CH
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
S
S
D001483:Base Sequence; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism*; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000201:enzymology*
D012330:RNA, Double-Stranded; D012313:RNA; D012316:RNA Nucleotidyltransferases; D020365:RNA Helicases