Using nucleotide analogs to probe protein-RNA interactions

Synthetic nucleotide analogs provide the opportunity to evaluate the importance of individual functional groups on the RNA in protein-RNA complexes. The general approach is to incorporate analogs at a defined position(s) in the RNA target and to evaluate the effect of this substitution on the thermodynamic stability of the protein-RNA complex. The underlying assumption is that if the presence of the analog reduces the stability of the complex, then the functional groups that are altered in the analog interact with the protein. Here we describe the protocols for incorporation of nucleotide analogs either by in vitro transcription using T7 RNA polymerase or by synthetic chemistry. We also describe how we have used this approach to study the interaction of the TRAP protein from Bacillus subtilis with its cognate RNAs consisting of 11 repeats of GAG and/or UAG triplets. By comparing the results 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 groups identified by analogs forge direct interactions with the protein. Analog studies also correctly identified residues that do not contact the protein. Moreover, analogs can have indirect effects on the complex stability by altering the structural properties of the RNA.