Our research is focused on RNA-processing proteins and RNA polymerase (RNAP)-associated transcription factors. We pioneered the structural analysis of dsRNA in complex with ribonuclease III (RNase III) enzymes. RNase III represents a family of dsRNA-specific endoribonucleases required for RNA maturation and gene regulation. Prokaryotic RNase III and eukaryotic Rnt1p, Dcr1, Drosha, and Dicer are representative members of the family. Previously, we reported a total of eleven crystal structures of a bacterial RNase III in complex with dsRNA at various catalytic stages of the enzyme, including the first structure of a catalytically meaningful RNase III-RNA complex and the structure of a catalytic stage immediately after the cleavage of the phosphodiester bond. Recently, we determined the crystal structure of a post-cleavage complex of Rnt1p from yeast, the first structure of a eukaryotic RNase III in complex with RNA in a catalytically meaningful manner. Strikingly, the structure features two rulers for substrate selection. This double-ruler mechanism represents an example of the evolution of substrate selectivity and provides a framework for understanding the catalytic mechanism of eukaryotic RNase IIIs. The worldwide effort in structural analysis of other eukaryotic RNase III enzymes resulted in several important structures, including the structures of Dicer, Dcr1, and Drosha. These structures, however, do not contain RNA and thus are not able to explain their mechanisms of action. Our structures of RNase III:dsRNA complexes greatly enhanced the significance of these important structures. Based on the protein-RNA interactions revealed by our structures of both prokaryotic and eukaryotic enzymes, models with RNA can be reliably constructed for Dicer, Dcr1, and Drosha. A model complex of Dicer with RNA explains how Dicer enzymes recognize the 2-nucleotide 3' overhang of dsRNA substrate and measure 22 nucleotides up to position the scissile bond over the cleavage site. A model complex of Dcr1 with RNA explains how homodimers of non-canonical Dicer enzymes bind cooperatively along dsRNA substrate such that the distance between active centers in adjacent homodimers is the length of 22 nt. A model complex of Drosha with RNA explains how Drosha enzymes recognize the last base pair in the basal junction of the primary microRNA substrate and measure 11 nucleotides up to position the scissile bond over the cleavage site. Our structural and mechanistic studies of biomolecular systems aim to reveal their reaction coordinates or functional cycle. To date, we have described the reaction coordinates of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK, a folate pathway enzyme essential for microorganisms but absent in mammals), the functional cycle of Era (an essential GTPase that couples cell growth with cell division), RapA (a Swi2/Snf2 protein that recycles RNA polymerase), bacterial RNase III, and yeast RNase III. Several biomolecular systems mentioned above are attractive molecular targets and structure-based drug development is an integral part of our research.
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