Structure of capping enzyme bound to the CTD from RNA polymerase II
Structure of the human DcpS dimer bound to RNA cap analog
Structure of Rnl2 bound to a nicked adenylated substrate
Our interest in RNA processing also extends to RNA repair. In collaboration with the Shuman laboratory, we have addressed the structural basis for RNA repair through the structure determination of RNA ligase intermediates.
We have described the structural and genetic basis for the 3 essential steps in cap formation.
i.) The triphosphatase enzyme catalyzes the gamma phosphate hydrolysis from the nascent 5’ triphosphate mRNA end;
ii.) the guanylyltransferase capped the 5’ diphosphate terminated mRNA in the next step; and
iii.) the final essential step involved methylation at the N7 guanine position.
We are actively engaged in understanding the structural basis for catalysis in this system as well as the structural basis for association of the capping apparatus on RNA polymerase II.
Eukaryotic messenger RNA degradation plays a critical role in regulating the turnover of RNA in the cell. In eukaryotic cells, 2 major pathways are utilized to degrade mRNA and both are initiated with deadenylation of the polyadenylated (poly[A]) tail. In the 5’ to 3’ decay pathway, the mRNA cap is hydrolyzed following deadenylation, exposing the 5’ end to 5’ to 3’ exoriboribonuclease activities. In the 3’ to 5’ decay pathway, degradation of the mRNA body continues from the 3’ end following deadenylation to generate a cap structure, which is subsequently hydrolyzed. We have determined the structural and biochemical basis for RNA decapping in the 3’ to 5’ pathway, and continue to work on aspects of the 5’ to 3’ pathway and exosome mediated RNA degradation.
T4 RNA ligase 2 (Rnl2) and kinetoplastid RNA editing ligases exemplify a family of RNA repair enzymes that seal 3’OH/5’PO(4) nicks in duplex RNAs via ligase adenylylation, AMP transfer to the nick 5’PO(4), and attack by the nick 3’OH on the 5’-adenylylated strand to form a phosphodiester. We have determined crystal structures for Rnl2 at discrete steps along this pathway, illuminating the stereochemistry of nucleotidyl transfer and revealing how remodeling of active-site contacts and conformational changes propel the ligation reaction forward. By comparing structures of Rnl2 and human DNA ligase I, we were able to highlight common and divergent themes of substrate recognition that explain their specialization for RNA versus DNA repair.