ABSTRACT
Catalytic RNAs or ribozymes are polynucleotides capable of carrying out a chemical reaction. In their natural context, most ribozymes act on themselves in an autolytic manner. RNase P, a ribozyme that processes pre-tRNAs, is a notable exception in that its natural behavior is cata lytic [1,2]. By engineering the sequence appropriately, most ribozymes have been converted into true catalysts in vitro, capable of acting on an independent substrate molecule [3]. Ribozymes vary greatly in their size and complexity. The smallest reported ribozyme is just three nu cleotides in length [4], whereas other ribozymes have minimal se quences hundreds of nucleotides long. Several of the more commonly studied small ribozymes (^100 nt) include hammerhead ribozyme, hep atitis 8 virus ribozyme, hairpin ribozyme, and Neurospora VS ribozyme. The class of large ribozymes includes group I intron, group II intron, and RNase P. Reviews of general ribozyme biochemistry are available in a number of recent sources [5-8] and will not be discussed in detail here. Instead, this chapter will focus on the experiments that use the special properties of Mn2+ as a probe for the Mg2+ binding sites on ribozymes. These methods include phosphorothioate rescue, X-ray crys tallography, electron paramagnetic resonance (EPR), and nuclear mag netic resonance (NMR). Most of the examples cited will revolve around
the hammerhead ribozyme, as this small catalytic motif has been stud ied extensively by these methods. This system shows the utility of these techniques as well as their limitations.
