Facilitation of hammerhead ribozyme catalysis by the nucleocapsid protein of HIV-1 and the heterogeneous nuclear ribonucleoprotein A1.
In order to improve the activity of hammerhead ribozymes in vivo, we have analyzed the effect of several prototypical RNA binding proteins on the ribozyme cleavage reaction: bacteriophage T4 gene 32 protein (gp32), hnRNP A1 (A1) and the nucleocapsid protein of HIV-1 (NCp7). We show that, while gp32 has no effect on the cleavage reaction, A1 and NCp7 affect different steps of the reaction. Moreover, some of these effects depend upon the ribozyme-substrate hybrid length. A1 and NCp7 inhibit the reaction of the least stable ribozyme-substrate complexes, which have 12 bp of duplex. NCp7, but not A1, inhibits the cleavage of substrates that have long ribozyme-substrate duplexes (17 or 20 bp), while cleavage of complexes having shorter duplexes (13 or 14 bp) is not affected. NCp7 and A1 enhance the turnover of ribozymes by increasing the rate of product dissociation, but only when both cleavage products are bound with < or = 7 bp. A1 and NCp7 enhance ribozyme binding to long substrates, such as mRNAs, the structure of which otherwise limits ribozyme binding. Therefore, the effects of A1 or NCp7 on the different steps of the cleavage reaction define a length of the ribozyme-substrate duplex which allows enhancement of the rate of binding and product release without inhibiting the cleavage step. Interestingly, this duplex length (14 bases, or 7 on each side of the cleavage site) is identical for A1 and NCp7. Since A1 is thought to interact with most, if not all mRNAs in vivo, it may enhance the intracellular activity of ribozymes targeted against any mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Bertrand
E L
EL
Beckman Research Institute of the City of Hope, Department of Molecular Genetics, Duarte, CA 91010.
Rossi
J J
JJ
D001483:Base Sequence; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins; D002384:Catalysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015683:Gene Products, gag / Q000378:metabolism*; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D016337:RNA, Catalytic / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D015683:Gene Products, gag; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C071884:NCP7 protein, Human immunodeficiency virus 1; D016337:RNA, Catalytic; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C060035:gp32 protein, Enterobacteria phage T4; C000619656:hnRNPA1 protein, human