In vitro NCp7 enhancement of ribozyme-mediated cleavage of full-length human IL-6 mRNA.
We have previously shown that a ribozyme directed against human interleukin-6 (IL-6) mRNA is efficient in vivo, despite its poor activity in vitro on full-length IL-6 mRNA. We compared the effect of the nucleocapsid protein of HIV-1 (NCp7) on the ribozyme cleavage reaction of a long (1041 nt) and a short (19 nt) substrate IL-6 RNA in vitro. At a one to five molar ratio of the long substrate to ribozyme, almost no cleavage is observed after 30 min at 37 degrees C. The NCp7 protein significantly increases the catalytic activity of the ribozyme on this substrate (from 0 to 53% after 7 min at 37 degrees C), but not on the short one. A kinetic analysis of single turnover reactions performed with ribozyme in at least fivefold molar excess over substrate also lead to a stimulation (70-fold) of the reaction rate with long substrate, but not with the shorter one. Preferential increases of the catalytic activity on the long substrate suggests that the NCp7 protein prevents misfolding of RNAs.
Mahieu
M
M
Institut Pasteur, Department of Virology, Bruxelles, Belgium.
Hendrix
C
C
Ooms
J
J
Herdewijn
P
P
Content
J
J
D001483:Base Sequence; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins; D015683:Gene Products, gag / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D015850:Interleukin-6 / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; D015850:Interleukin-6; C071884:NCP7 protein, Human immunodeficiency virus 1; D016337:RNA, Catalytic; D012333:RNA, Messenger; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus