Application of hammerhead ribozymes for structural studies of ribosomal 5S RNAs

We synthesized and used hammerhead ribozymes designed for the GUC and AUC target sequences located in the terminal loop C of animal and plant 5S rRNAs to verify its involvement in a higher order structure formation of ribosomal RNA. The activity of both GUC and AUC specific ribozymes were proven to be active against synthetic analogs of the stem III and loop C in the two types of 5S rRNAs. In contrast we did not observe any ribozyme-catalysed hydrolysis within the loop C of the native 5S rRNAs. We considered two possible explanations of the results: an involvement of the loop C in long range interactions and lack of a structural flexibility to form proper tertiary structure of the complex with the ribozyme. From our data it seems that the single stranded nucleotides within the loop C can not form correct RNA duplex structure required for ribozyme activity. It is clear that hammerhead ribozymes can be used for inhibition of different steps of expression of genetic information only if the target sequence occurs in appropriate context, that allows to form correct secondary and tertiary structure of the ribozyme-substrate complex.