Isolation and Characterization of Ribozymes Catalyzing Aminoacylation from CoA Thioesters
Date of Award
Doctor of Philosophy (PhD)
Chemistry and Biochemistry
Chemistry and Biochemistry
The RNA world hypothesis suggests that current DNA and protein dominant world is preceded by a simple life form prominently based on RNA. It is supported by the discovery of naturally occurring ribozymes and the isolation of a variety of ribozymes by in vitro selection . Coenzyme A thioesters play essential role in current metabolism. To demonstrate plausible biochemical functions of thioesters in the RNA world, we have isolated a new class of ribozymes that catalyze efficient self-aminoacylation using aminoacyl CoA. We hypothesize that ribozyme-based aminoacylation systems using aminoacyl thioesters of CoA could have existed as the ancestors of current aminoacyl tRNA synthetases. Combining with our previous results (CoA and thioester synthesis by RNA), extensive RNA-catalyzed metabolic pathways involving CoA and its thioesters is proposed. Complex contemporary metabolic systems could have evolved from the proposed ribozyme pathways. An isolated ribozyme (ACT3) not only can catalyze the efficient synthesis of aminoacylated RNA (forward reaction) from aminoacyl CoA thioesters, but is also capable of making CoA thioesters from aminoacylated RNA and free CoA (reverse reaction). Furthermore, we have discovered an exquisite CoA binding site within ACT3. Using different CoA analogs, we have been able to map the chemical moiety of CoA that is responsible for the CoA-RNA binding---with 3' AMP being the major binding determinant and the 5' phosphate making an additional contribution. The equilibrium constant for the ribozyme catalyzed reaction is close to 1. However, the equilibrium constant calculated from the difference of the Gibbs free energy between thiol esters hydrolysis and oxygen esters hydrolysis is approximately 70 favoring the formation of oxygen esters. The ribozyme folds in such a way that the local environment of functional hydroxyl group of U50 has been changed and we hypothesize that free energy of hydrolysis of formed ester bond is increased thereby the equilibrium constant of ACT3-tr1 catalyzed reaction is affected. Investigation of RNA structure/function/mechanism frequently requires the utilization of 5' labeled RNA. A novel method to label RNA at its 5' end with fluorescence through one-step transcription has been developed in our laboratory. We have successfully labeled ACT3 with this technique and determined its reaction site utilizing the 5' fluorescence-ACT3.
Li, Na, "Isolation and Characterization of Ribozymes Catalyzing Aminoacylation from CoA Thioesters" (2006). Dissertation Archive. 479.