Nuclease footprint analyses of the interactions between RNase P ribozyme and a model mRNA substrate.

AUTOR(ES)

Trang, P

RESUMO

RNase P ribozyme cleaves an RNA helix substrate which resembles the acceptor stem and T-stem structures of its natural tRNA substrate. By linking the ribozyme covalently to a sequence (guide sequence) complementary to a target RNA, the catalytic RNA can be converted into a sequence-specific ribozyme, M1GS RNA. We have previously shown that M1GS RNA can efficiently cleave the mRNA sequence encoding thymidine kinase (TK) of herpes simplex virus 1. In this study, a footprint procedure using different nucleases was carried out to map the regions of a M1GS ribozyme that potentially interact with the TK mRNA substrate. The ribozyme regions that are protected from nuclease degradation in the presence of the TK mRNA substrate include those that interact with the acceptor stem and T-stem, the 3' terminal CCA sequence and the cleavage site of a tRNA substrate. However, some of the protected regions (e.g. P13 and P14) are unique and not among those protected in the presence of a tRNA substrate. Identification of the regions that interact with a mRNA substrate will allow us to study how M1GS RNA recognizes a mRNA substrate and facilitate the development of mRNA-cleaving ribozymes for gene-targeting applications.

Documentos Relacionados

• Differential effects of the protein cofactor on the interactions between an RNase P ribozyme and its target mRNA substrate
• Base pairing between Escherichia coli RNase P RNA and its substrate.
• RNase MRP and RNase P share a common substrate.
• Requirements for cleavage by a modified RNase P of a small model substrate.
• Targeted cleavage of mRNA by human RNase P.