Biomolecular Structure and Mechanism, Structure-Based Drug Design
Our research is focused on the structural biology of RNA biogenesis, with an emphasis on RNA-processing proteins and RNA polymerase-associated transcription factors, and structure-based development of therapeutic agents. The goal of structural analysis is to map the reaction trajectory or functional cycle of selected biological macromolecules, and that of drug discovery is to design, synthesize, and characterize novel anticancer and antimicrobial agents. To date, we have described the reaction trajectory and/or functional cycle of HPPK (a folate pathway enzyme essential for microorganisms but absent in mammals), Era (an essential GTPase that couples cell growth with cell division), RapA (a Swi2/Snf2 protein that recycles RNA polymerase), and bacterial and yeast RNase III enzymes. Please see our Gallery for the structural view of these biological macromolecules and their reaction trajectory or functional cycle. Our contributions in RNase III research are summarized below, followed by examples of structure-based design of anticancer and antimicrobial agents.
Structural view of RNA processing by RNase III enzymes. Representative members of the RNase III family include prokaryotic RNase III and eukaryotic Rnt1p, Drosha, and Dicer. They play important roles in RNA processing and maturation, post-transcriptional gene silencing, and defense against viral infection. For structural and mechanistic studies, bacterial RNase III and yeast Rnt1p are valuable model systems for prokaryotes and eukaryotes, respectively. For both RNase III and Rnt1p, we have shown how the dimerization of their endonuclease domain (RIIID) creates a catalytic valley where two cleavage sites are located, how the catalytic valley accommodates a dsRNA substrate in a manner such that each of the two RNA strands is aligned with one of the two cleavage sites, how the hydrolysis of each strand involves both RIIIDs, and how RNase III uses the two cleavage sites to create 2-nucleotide (2-nt) 3' overhangs in its products. We have also shown how magnesium is essential for the formation of a catalytically competent protein-RNA complex, and how the use of magnesium ions can drive the hydrolysis of each phosphodiester bond. Moreover, we have described a stepwise model by which RNase III and Rnt1p execute the phosphoryl transfer reaction. All members of the RNase III family propel RNA hydrolysis by two-Mg2+-ion catalysis, which exhibits distinct features, however, by prokaryotes and eukaryotes. As revealed by our structures, prokaryotic RNase IIIs require a third magnesium ion in catalysis, whereas eukaryotic RNase IIIs employ two additional amino acid side chains. For further details on bacterial RNase III and yeast Rnt1p, please see our review articles (PMID: 24274754; PMID: 30548404) and a recent report (PMID: 35829618).
Based on the protein-RNA interactions revealed by our structures of RNase III and Rnt1p, models with RNA can be meaningfully constructed for both Drosha and Dicer. A model complex of Drosha with RNA explains how Drosha enzymes recognize the last base pair in the basal junction of a primary microRNA substrate and measure 11 nucleotides up to position the scissile bond over the cleavage site. A model complex of Dicer with RNA explains how Dicer enzymes recognize the 2-nt 3' overhang of a dsRNA substrate and measure 22 nucleotides up to position the scissile bond over the cleavage site.
To fully characterize substrate specificity and product size of bacterial RNase III enzymes, we performed in vitro cleavage of dsRNAs by Ec and Aquifex aeolicus (Aa) RNase IIIs and delineated their products by next generation sequencing. Surprisingly, we found that both enzymes cleave dsRNA at preferred sites, among which a guanine nucleotide was enriched at a specific position (+3G). Based on sequence and structure analyses, we conclude that RNase IIIs recognize +3G via a conserved glutamine (EcQ165/AaQ161) side chain. Abolishing this interaction by mutating the glutamine to an alanine eliminates the observed +3G preference. Based on this finding, we created a research tool and named it bacterial Dicer that is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies (PMID: 30916338).
Structure-based Development of Anticancer and Antimicrobial Agents. We carry out structure-based drug development primarily as a continuation of our basic research on the structure and mechanism of biomolecular systems with anticancer and antimicrobial significance. Previously, we designed PABA/NO, an enzymatically activated anticancer prodrug that kills cancer cells from within by releasing nitric oxide. We also made significant progress toward novel antibacterial agents targeting HPPK by the design and synthesis of linked purine pterin inhibitors and the determination of their crystal structures in complex with the enzyme. Recently, we have further developed both the PABA/NO and HPPK inhibitors. The PABA/NO derivative contains a PARP inhibitor and so it not only generates nitric oxide but also release a PARP inhibitor simultaneously in the same compartment, which damage DNA and inhibit its repair (PMID: 24521039; US Patent Number: 9168266). The improved HPPK inhibitors mimic closely the transition state of the catalytic complex and thereby have much higher potency (PMID: 33199204; US Patent Number: 11091509).
Dr. Ji earned his Ph.D. degree at the University of Oklahoma (1985-1990) and performed his postdoctoral research at the University of Maryland (1991-1994), where he became a Research Assistant Professor before joining the National Cancer Institute (NCI), National Institutes of Health (NIH). At the NCI-Frederick, Dr. Ji established his laboratory in the ABL-Basic Research Program in 1995, moved to the Center for Cancer Research in 1999, and gained tenure as an NIH Senior Investigator in 2001. He is a member of American Crystallographic Association (1986-present), the Editor of the Newsletter of NIH X-ray Diffraction Interest Group (2001-present), and a member of the Steering Committee of NCI RNA Biology Initiative (2018-present).
- Song H, Fang X, Jin L, Shaw GX, Wang YX, Ji X. The Functional Cycle of Rnt1p: Five Consecutive Steps of Double-Stranded RNA Processing by a Eukaryotic RNase III. Structure. 2017;25(2):353-363.
- Liang YH, Lavoie M, Comeau MA, Abou Elela S, Ji X. Structure of a eukaryotic RNase III postcleavage complex reveals a double-ruler mechanism for substrate selection. Mol Cell. 2014;54(3):431-44.
- Court DL, Gan J, Liang YH, Shaw GX, Tropea JE, Costantino N, Waugh DS, Ji X. RNase III: Genetics and function; structure and mechanism. Annu Rev Genet. 2013;47:405-31.
- Stagno JR, Ma B, Li J, Altieri AS, Byrd RA, Ji X. Crystal structure of a plectonemic RNA supercoil. Nat Commun. 2012;3:901.
- Gan J, Tropea JE, Austin BP, Court DL, Waugh DS, Ji X. Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III. Cell. 2006;124(2):355-66.
Related Scientific Focus Areas
Microbiology and Infectious Diseases
Molecular Biology and Biochemistry
This page was last updated on Wednesday, August 30, 2023