NTPase and RNA helicase activities of the hepatitis C Virus NS3 protein = C형 간염 바이러스 NS3 단백질의 NTPase, RNA helicase 활성에 관한 연구

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Hepatitis C Virus (HCV) is the major etiological agent of non-A, non-B post-transfusion hepatitis. Its genome, a (+) stranded RNA molecule of about 9.4 kb, encodes a large polyprotein that is processed by viral and cellular proteases into at least nine different viral polypeptides. NS3 (non-structural) protein has a sequence motifs of a serine protease at N-terminal 1/3 fragment, and a sequence motifs of a NTPase/DEXH RNA helicase at C-terminal 2/3 fragment. The carboxy terminal 3/4 fragment (466 a.a) of the HCV is expressed in E.coli with T7 promoter and His-tag purification system and designated as NS3H. NS3H has a polynucleotide stimulated NTPase activity, and an RNA helicase activity. The NS3H requires ATP and divalent cations such as $Mg^{2+}$ and $Mn^{2+}$ for RNA helicase activity. NS3H is able to utilize any nucleotide for RNA helicase activity, and it shows optimum activity at 3 mM divalent ion, and pH 6.5. The minimal size for the full enzymatic activity of the NS3 protein is 400 amino acid fragment which is deleted 16 amino acids from N-terminus, and 50 amino acids from C-terminus of NS3H. The contribution of the individual residues in conserved sequence motifs of NS3H which is a member of DEXH RNA helicase is investigated by mutational analysis and biochemical assays. Lysine residue in AXXXXGKS plays important role in NTPase activity and in coupling the NTPase activity with the RNA helicase activity. Not all the amino acids can occupy the X residue in DEXH motif of HCV NS3 RNA helicase. At least serine cannot be X residue. NTPase activity of the NS3H could be increased by substitution of histidine with alanine, and this implies that the DEXH motif is crucial for NTPase activity. TAT motif is involved in RNA unwinding activity of the NS3H The QRRGRTGR motif is not important in RNA binding but, is involved in NTP hydrolysis and consequently in RNA unwinding activity. The Gln residue, the second, third and forth arginine residues are indispensable for ...
Advisors
Choe, Joon-Horesearcher최준호researcher
Description
한국과학기술원 : 생물과학과,
Publisher
한국과학기술원
Issue Date
1997
Identifier
112591/325007 / 000935039
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생물과학과, 1997.2, [ xi, 112 p. ]

Keywords

RNA helicase; Deletion; Point mutation; C형 간염 바이러스; 엔티피에이즈; 알엔에이 나선효소; 결실; 치환 돌연변이; Hepatitis C virus; NTPase

URI
http://hdl.handle.net/10203/27400
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=112591&flag=dissertation
Appears in Collection
BS-Theses_Ph.D.(박사논문)
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