A Double-Site Acting Kinase Ribozyme
E. Biondi and D.H. Burke
Department of Molecular Microbiology and Department of Biochemistry, University of Missouri-Columbia
Our long-term goal is to understand the catalytic potential of RNA, the feasibility of RNA-based evolution in an RNA World, and the possibility of using RNA to engineer artificial gene regulation and metabolism. A key constraint in the acquisition of new biochemical functions is the ability of a ribozyme to accommodate diffusible substrates. We are analyzing the mechanism and catalytic requirements of kinase ribozymes. RNA-catlyzed phosphorylations are attractive to study for several reasons. Among them, phosphoryl transfer is one of the most important and ubiquitous reactions in any type of metabolism, and of fundamental biological and evolutionary significance. The present work describes a kinase ribozyme, MK28-2, which was selected to use GTP(S) as (thio)phosphoryl donor. MK28-2 promotes (thio) phosphorylation of two distinct 2 hydroxyls that are widely separated in primary sequence. The secondary structure of the active molecule was obtained by enzymatic probing and nucleotide requirements for catalysis and tertiary interactions identified by mutational analysis. The path and fate of double (thio)phosphorylation was followed with the aim of understanding the catalytic potential of the active site. Metal requirements for optimal activity were also investigated, showing a high dependence of the ribozyme on magnesium and copper ions. Moreover, this RNA was shown to be the first kinase ribozyme which activity is influenced by pH variations. The results obtained with these experiments provide new prospects on the mechanism of catalysis and substrate binding demand for kinase ribozymes. These studies will help delineate the catalytic potential of metabolic ribozymes in the contexts of both an RNA World and an RNA-based engineered metabolism.