Dr. Ji pioneered the structural analysis of double-stranded RNA (dsRNA) in complex with RNase III enzymes. Over the past decade, he has provided the structural view of dsRNA processing by bacterial RNase III. Recently, his structure of a eukaryotic RNase III has revealed a picking motif in structured RNA and a double-ruler mechanism for substrate selection.
As Head, Dr. Ji directs the Section’s basic and translational research program that is focused on the structural biology of RNA biogenesis, especially post-transcriptional RNA processing, and structure-based development of anticancer and antimicrobial agents.
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 for structural analysis is to map the reaction trajectory or functional cycle of selected biological macromolecules, and that for drug development is to design, synthesize, and characterize novel anticancer and antimicrobial agents. See Research Gallery and Drug Discovery Patents for the scope and depth of our science. Examples of biomolecular systems under investigation are described below.
RNase III (ribonuclease III) enzymes, exemplified by bacterial RNase III and eukaryotic Rnt1p, Drosha, and Dicer, play important roles in RNA processing and maturation, post-transcriptional gene silencing, and defense against viral infection. For mechanistic studies, the bacterial enzyme is a valuable model system for the entire family. We have shown how the dimerization of the endonuclease domain of RNase III creates a catalytic valley where two cleavage sites are located, how the catalytic valley accommodates a dsRNA 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 subunits, and how RNase III uses the two cleavage sites to create the 2-nucleotide 3' overhangs in its products (View). We have also shown how magnesium is essential for the formation of a catalytically competent protein-RNA complex, how the use of two magnesium ions can drive the hydrolysis of each phosphodiester bond, and how conformational changes in both the substrate and the protein are critical elements for assembling the catalytic complex. Moreover, we have provided a stepwise mechanism for the enzyme to execute the phosphoryl transfer reaction (View). As informative as the bacterial enzyme for the mechanism of RNase III action, yeast Rnt1p is a valuable model system for eukaryotic RNase III enzymes. Unlike bacterial enzyme that uses four catalytic side chains, eukaryotic RNase III uses six. Also distinguished from the bacterial enzyme, every eukaryotic RNase III has an N-terminal extension. What is more, Rnt1p exhibits a strict guanine nucleotide specificity, which is unique among RNase III enzymes. We have shown how the substrate-binding mode of Rnt1p is distinct from that of bacterial RNase III (View), how all of the six catalytic side chains are engaged in the cleavage site (View), how a new RNA-binding motif of Rnt1p functions as a guanine-specific clamp (View), and how the double-stranded RNA-binding domain and N-terminal domain of Rnt1p function as two rulers measuring the distance between the guanine nucleotide to the cleavage sites (View). This unusual mechanism of substrate selectivity represents an example of the evolution of substrate selectivity and provides a framework for understanding the mechanism of action of other eukaryotic RNase III enzymes, including Drosha, and Dicer.
HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) catalyzes magnesium-dependent pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). The reaction follows a bi-bi mechanism with ATP as the first substrate and AMP and HP pyrophosphate (HPPP) as the two product molecules. HPPK is a key enzyme in the folate biosynthetic pathway. It is essential for microorganisms but absent from mammals. Unlike dihydropteroate synthase (another key enzyme in the folate pathway), which is the target of sulfonamides (the first widely used synthetic antibiotics), HPPK is not the target for any existing antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. We have constructed a reaction coordinate on the basis of thermodynamic and transient kinetic data and mapped out the reaction trajectory by determining five crystal structures of the enzyme at various liganded states (View). On the basis of structural and mechanistic information, we have been attempting the development of HPPK inhibitors as novel antimicrobial agents (View).
Selected Key Publications
Structure of a eukaryotic RNase III postcleavage complex reveals a double-ruler mechanism for substrate selection.Mol. Cell. 54: 431-444, 2014. [ Journal Article ]
- Nat Commun. 3: 901, 2012. [ Journal Article ]
- Proc. Natl. Acad. Sci. USA. 108: 10156-10161, 2011. [ Journal Article ]
- Structure. 16: 1417-1427, 2008. [ Journal Article ]
- Cell. 124: 355-66, 2006. [ Journal Article ]
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 (1994-1995) before joining the National Cancer Institute (NCI), National Institutes of Health (NIH). At the NCI at Frederick, Dr. Ji established his laboratory in the ABL-Basic Research Program in 1995, moved to the Center for Cancer Research in 1999, gained tenure in 2001 as an NIH Senior Investigator, and in 2008 became a member of the Senior Biomedical Research Service (SBRS). The SBRS, established under the Public Health Service Act, was created for scientists who are considered by their peers to be outstanding in their work.
|Lan Jin Ph.D.||Research Fellow|
|Vandana Kumari Ph.D.||Postdoctoral Fellow (Visiting)|
|Gary Shaw Ph.D.||Research Biologist|
|Genbin Shi, Ph.D.||Staff Scientist|
|He Song Ph.D.||Postdoctoral Fellow (Visiting)|
|Chao Wang Ph.D.||Postdoctoral Fellow (Visting)|
Dr. Smita Kakar
- Recipient of the Poster Award at the NCI Structural Biology Retreat for outstanding presentaton of her work as shown below (2015).
- Dr. Smita Kakar was a WINNER of the NIH Fellows Award for Research Excellence (FARE) 2016 competition for her work entitled “Allosteric Activation of Bacterial Swi2/Snf2 Protein RapA by RNA Polymerase: Biochemical and Structural Studies” (2015).
Dr. Yu-He Liang
- Dr. Liang’s paper entitled “Structure of a eukaryotic RNase III post-cleavage complex reveals a double-ruler mechanism for substrate selection” was featured on the issue’s cover of Molecular Cell (2014).
- Selected to receive The Protein Society Young Investigator Award, Protein Society Young Investigator Travel Grant, and The Protein Society Finn Wold Travel Award with an invited talk entitled “Structural Basis for Sequence Specificity and Product Length of Yeast Ribonuclease III” (2012).
Dr. Jason Stagno
- WINNER of the NIH Fellows Award for Research Excellence (FARE) 2013 competition for his work entitled “Structural Basis for NusB in the Initiation of Transcription Antitermination and dsRNA Supercoiling” (2012).
- Selected to receive The American Crystallographic Association Travel Award with an invited talk entitled “Crystal structure of a plectonemic RNA supercoil” (2012).
- Recipient of The SER-CAT Young Investigator Award for his paper entitled “Structural basis for RNA recognition by NusB and NusE in the initiation of transcription antitermination” (2012).
Dr. Chao Tu
- WINNER of the NIH Fellows Award for Research Excellence (FARE) 2010 competition for his work entitled “Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis” (2009).
- Winner of The Outstanding Scientific Presentation Award for his talk entitled “ERA: a GTP-dependent Molecular Switch Recognizes the 3’ End of 16S rRNA” in the Chemistry as a Life Science Symposium at the National Cancer Institute’s Spring Research Festival at Frederick (2009).
- Selected to receive The Protein Society Young Investigator Award, Protein Society Young Investigator Travel Grant, and The Protein Society Finn Wold Travel Award with an invited talk entitled “Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis” (2009).
Dr. Jianhua Gan
- Selected as the recipient of The SER-CAT Young Investigator Award for his paper entitled “Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III” (2006).
- WINNER of the NIH Fellows Award for Research Excellence (FARE) 2007 competition for his work entitled "The Mechanism of Double-Stranded RNA Cleavage by Ribonuclease III: How Dicer Dices” (2006).
Dr. Bing Xiao
- Figure 3 in Dr. Bing Xiao’s paper entitled “Crystal structure of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, a potential target for the development of novel antimicrobial agents” has been reproduced in the college textbook BIOCHEMISTRY, the 3rd and all subsequent editions, by Donald Voet and Judith Voet (2002).
Dr. Jaroslaw Blaszczyk
- Dr. Blaszczyk’s paper entitled “Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage” was featured on the issue’s cover of Structure (2001).