Structure-function relationships in enzymes of the nucleotidyltransferase superfamily: RNA ligation and mRNA capping.

摘要

The nucleotidyltransferase superfamily is composed of ATP and NAD + dependent DNA ligases, ATP-dependent RNA ligases and GTP dependent mRNA capping enzymes. Despite a wealth of mutational analysis and structural data, few studies characterize the functional dynamics of these enzymes. Consequently, it is the goal of this dissertation to remedy, at least in part, that short coming. Specifically, we use molecular dynamic methodologies to model dynamic behavior of the ATP-dependent RNA editing ligase, TbREL1, from Trypanosoma brucei, the causative agent of African sleeping sickness, and the GTP-dependent mRNA capping enzyme from the Paramecium bursaria Chlorella virus, PBCV-1. Chapter two investigates the allosteric and local effects of ATP binding in the active site of TbREL1 and predicts the binding mode of nicked dsRNA substrate by calculating ensemble averaged electrostatic potentials. Chapter three discusses the functional dynamics of conserved active-site residues that may be important in mediating nicked double stranded RNA ligation, predicts that TbREL1 requires two magnesium ions for optimal catalysis, and provides insight into the experimental observation that TbREL1 requires that the 5´-phosphate nicked strand be RNA. Chapter four describes the use of targeted molecular dynamics to explore the role of conserved residues in mediating a catalytically requisite substrate isomerization event in the PBCV-1 mRNA capping enzyme, discussing the modeled dynamics in context of previously reported mutational analysis. Chapter five examines the impact that the rate and extent of OB domain motion has on various aspects of GTP dependent PBCV-1 mRNA capping enzyme function. Specifically, the rate of OB domain motion is discussed in terms of its effect on the GTP association rate, while the extent of motion provides the context to re-examine a putative mechanism explaining the ability of the enzyme to selectively bind single-stranded mRNA. Furthermore, chapter five discusses the effects of GTP binding in context of the induced fit and population shift models of substrate binding.