CRYSTALLIZATION OF RNA-PROTEIN COMPLEXES .1. METHODS FOR THE LARGE-SCALE PREPARATION OF RNA SUITABLE FOR CRYSTALLOGRAPHIC STUDIES

In vitro transcription using bacteriophage RNA polymerases and lineari sed plasmid or oligodeoxynucleotide templates has been used extensivel y to produce RNA for biochemical studies. This method is, however, not ideal for generating RNA for crystallisation because efficient synthe sis requires the RNA to have a purine rich sequence at the 5' terminus , also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virt ually any sequence for crystallization. In the first method RNA is tra nscribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary trans cript, eliminating the promoter sequence and heterogeneous 3' end gene rated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme acti vity generates few sequence restrictions at the 3' terminus and none a t the 5' terminus, a considerable improvement on current methodologies . In the second method the BsmAI restriction endonuclease is used to l inearize plasmid template DNA thereby allowing the generation of RNA w ith any 3' end. In combination with a 5' cis-acting hammerhead ribozym e any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs.