Preparation of 5S RNA-Related Materials for Nuclear Magnetic Resonance and Crystallography Studies

This chapter describes the techniques used for purifying and manipulating 5S RNA and its various nucleolytic fragments, taking advantage of the availability of 5S RNA-overproducing strains. All the preparations described yield nuclear magnetic resonance (NMR)-scale quantities of end product. The fact that NMR is nondestructive, and that samples can be reused is helpful, but the need for large amounts of material remains. In this context, bacterial strains capable of overproducing the materials of interest are more than a mere convenience. The preparation of 5S RNA from chloramphenicol-treated pKK5-1- containing cells is straightforward. The cells are broken open; any of the standard methods will do. The ribosomes and cell debris are removed by centrifugation, and the RNA in the supernatant is isolated by phenol extraction. The 5S component of the resulting RNA mixture is purified by chromatography.