Using nucleotide analogs to probe protein-RNA interactions

Synthetic nucleotide analogs provide the opportunity to evaluate the import ance of individual functional groups on the RNA in protein-RNA complexes. T he general approach is to incorporate analogs at a defined position(s) in t he RNA target and to evaluate the effect of this substitution on the thermo dynamic stability of the protein-RNA complex. The underlying assumption is that if the presence of the analog reduces the stability of the complex, th en the functional groups that are altered in the analog interact with the p rotein. Here we describe the protocols for incorporation of nucleotide anal ogs either by in vitro transcription using T7 RNA polymerase or by syntheti c chemistry. We also describe how we have used this approach to study the i nteraction of the TRAP protein from Bacillus subtilis with its cognate RNAs consisting of 11 repeats of GAG and/or UAG triplets. By comparing the resu lts of these analog studies with the crystal structure of TRAP bound to an RNA containing 11 GAG repeats, we are able to see that all the functional g roups identified by analogs forge direct interactions with the protein. Ana log studies also correctly identified residues that do not contact the prot ein. Moreover, analogs can have indirect effects on the complex stability b y altering the structural properties of the RNA. (C) 2001 Academic Press.