The broad objective of the proposed research is to understand mechanisms by which proteins can regulate RNA function by promoting RNA folding. The mechanisms by which proteins recognize their """"""""unfolded"""""""" RNA ligands and induce RNA folding remain significantly challenging questions. To address this issue, the principal investigator will focus on multifunctional mitochondrial (mt) protein from Aspergillus that promotes catalytic group I intron splicing (the COB intron) by inducing RNA folding. Amazingly, the protein, IAniI, also catalyzes site-specific DNA cleavage. The dual function of IAniI places interesting constraints on protein structure such that it must co-op existing rigid surfaces or utilizes an inherent structural plasticity to perform unrelated functions. The proposed research is intended to address fundamental questions related to RNA folding, strategies of nucleic acid recognition and the evolution of protein form and function.
Specific aims are (1) To identify RNA structural transitions that accompany IAniI facilitated splicing of the COB pre-RNA. Both equilibrium and time-resolved RNA structure mapping will reveal the structural basis for the inability of COB intron to splice in low Mg2+ and identify conformational changes promoted by IAniI as well as characterize major transitions in the RNA/protein assembly pathway. These studies will address how protein binding and RNA folding are integrated to produce a functional RNA conformation. (2) To identify the structural basis for the specificity of IAniI binding to RNA and DNA. Modification/interference, DNA footprinting and in vitro selection experiments will identify functional groups that mediate IAniI specificity which will be a first step in comparison of the RNA and DNA recognition strategies of IAniI. (3) Identification and analysis of the functional domains of IAniI. Site-specific RNA cross-linking, affinity cleavage and mutagenesis will be used o define IAniI domain structure and determine if the DNA and RNA functions of IAniI require separate o integrated domains. A collaboration with Dr. Stoddard of FHCR is established to use X-ray crystallography to solve the structure of IAniI bound to its substrates. A co-crystal of IAniI and its DNA target site diffracts to high resolution and the resulting structure will provide a powerful reference to establish structure-function relationships.
