title: NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork.
abstract: RNA helicases regulate virtually all RNA-dependent cellular processes. Although much is known about helicase structures, very little is known about how they deal with barriers in RNA and the factors that affect their processivity. The hepatitis C virus encodes NS3, an RNA helicase that is essential for viral RNA replication. We have used optical tweezers to determine at the single-molecule level how the local stability of the RNA substrate affects the enzyme rate of strand separation, whether separation occurs by an active or a passive mechanism, and whether processivity is affected. We show that sequence barriers in RNA modulate NS3 activity. NS3 processivity depends on barriers ahead of the opening fork. Our results rule out a model where NS3 passively waits for the thermal fraying of double-stranded RNA. Instead, we find that NS3 destabilizes the duplex before separating the strands. Failure to do so before a strong barrier leads to helicase dissociation and limits the processivity of the enzyme.
authors: QB3 Institute, University of California, Berkeley, CA 94720, USA.
authors: Wei
authors: W
authors: Cheng
authors: Sophie
authors: S
authors: Dumont
authors: Ignacio
authors: I
authors: Tinoco
authors: Carlos
authors: C
authors: Bustamante
mesh_terms: D001483:Base Sequence; D016174:Hepacivirus / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
references: Cell. 2001 Jan 26;104(2):177-90
references: 11207360
references: Methods Enzymol. 2003;361:134-62
references: 12624910
references: Proc Natl Acad Sci U S A. 2006 Apr 18;103(16):6190-5
references: 16606839
references: Phys Rev E Stat Nonlin Soft Matter Phys. 2005 Jan;71(1 Pt 1):011904
references: 15697627
references: Science. 1992 Nov 13;258(5085):1122-6
references: 1439819
references: Science. 2001 Apr 27;292(5517):733-7
references: 11326101
references: J Phys Condens Matter. 2005 Nov 30;17(47):S3851-69
references: 21690729
references: Nat Rev Mol Cell Biol. 2004 Mar;5(3):232-41
references: 14991003
references: Science. 1994 Sep 9;265(5178):1599-600
references: 8079175
references: Nucleic Acids Res. 2001 Jan 15;29(2):565-72
references: 11139627
references: J Mol Biol. 2004 Oct 8;343(1):101-14
references: 15381423
references: Biochemistry. 1993 Jul 13;32(27):6815-20
references: 8392863
references: Science. 2003 Aug 29;301(5637):1235-8
references: 12947199
references: Nature. 2000 Jan 27;403(6768):447-51
references: 10667799
references: J Mol Biol. 2002 Aug 30;321(5):807-19
references: 12206763
references: Nature. 2006 Jan 5;439(7072):105-8
references: 16397502
references: J Mol Biol. 2006 May 12;358(4):974-82
references: 16569413
references: J Mol Biol. 2004 Dec 10;344(5):1287-309
references: 15561144
references: Cell. 2005 Jan 14;120(1):49-58
references: 15652481
references: Biochemistry. 2001 Jul 31;40(30):8898-904
references: 11467951
references: Science. 2003 Mar 21;299(5614):1892-5
references: 12649482
references: Nature. 2004 Jul 22;430(6998):476-80
references: 15269774
references: Nat Struct Mol Biol. 2005 May;12(5):429-35
references: 15806107
references: Nat Rev Drug Discov. 2002 Nov;1(11):867-81
references: 12415247
references: Mol Cell. 2007 May 11;26(3):335-47
references: 17499041
references: Annu Rev Biochem. 1996;65:169-214
references: 8811178
references: Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8602-6
references: 9671724