Differential effects of the protein cofactor on the interactions between an RNase P ribozyme and its target mRNA substrate.
RNase P from Escherichia coli is a tRNA-processing enzyme and consists of a catalytic RNA subunit (M1 RNA) and a protein component (C5 protein). M1GS, a gene-targeting ribozyme derived from M1, can cleave a herpes simplex virus 1 mRNA efficiently in vitro and inhibit its expression effectively in viral-infected cells. In this study, the effects of C5 on the interactions between a M1GS ribozyme and a model mRNA substrate were investigated by site-specific UV crosslink mapping. In the presence of the protein cofactor, the ribozyme regions crosslinked to the substrate sequence 3' immediately to the cleavage site were similar to those found in the absence of C5. Meanwhile, some of the ribozyme regions (e.g. P12 and J11/12) that were crosslinked to the leader sequence 5' immediately to the cleavage site in the presence of C5 were different from those regions (e.g. P3 and P4) found in the absence of the protein cofactor and were not among those that are believed to interact with a tRNA. Understanding how C5 affects the specific interactions between the ribozyme and its target mRNA may facilitate the development of gene-targeting ribozymes that function effectively in vivo, in the presence of cellular proteins.
Hsu
A W
AW
Program in Infectious Diseases and Immunity and Program in Comparative Biochemistry, School of Public Health, University of California, Berkeley, CA 94720, USA.
Kilani
A F
AF
Liou
K
K
Lee
J
J
Liu
F
F
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins; D018259:Herpesvirus 1, Human / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D043262:Ribonuclease P; D013379:Substrate Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012367:RNA, Viral; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli