Structural organization and RNA-binding properties of the DNA polymerases of bacteriophages T4 and RB69
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Description
DNA polymerase of bacteriophage T4 is a single-chained multifunctional DNA-binding replication enzyme that also binds its own mRNA and represses its own translation in vivo. The work described in this dissertation was undertaken to (a) elucidate the structural organization of this protein, (b) identify its functional domains, and (c) localize its mRNA binding pocket We have cloned the DNA polymerase gene (gene 43) of phage RB69 (a distant relative of T4) and compared its biological, enzymological, and nucleic acid binding properties to those of the T4 homologue. The two enzymes exhibit similar catalytic (in vitro) and complementing (in vivo) activities; however, the T4 enzyme exhibits strong preference to its own genome. One unexpected outcome of this work was that RB69 gp43 proved to be crystallizable. Currently, the RB69 enzyme is the only $\alpha$-like DNA-dependent DNA replication polymerase for which structural information has been obtained. The structural information has guided the strategy of site-directed mutagenesis and the construction of T4-RB69 gp43 chimeras to test the structure-function relationships of this single-chained multifunctional enzyme Although autogenous translational regulation has been conserved during evolution of RB69 gp43, RNA-binding specificity of the RB69 protein is diverged from that of the T4 homologue. That is, T4 gp43 preferentially binds (in vitro) and represses (in vivo) the operator RNA from its own genome, whereas the RB69 protein can bind and repress both the T4 and RB69 operators. The RNA specificity differences between these two proteins are being used to map the gp43 RNA binding site. In studies with T4-RB69 gp43 chimeras, we mapped an RNA binding region of the protein to a segment that also harbors important determinants for the polymerase catalytic function. Our results suggest that RNA functions as a regulator of both the dosage and catalytic activities of this replicative DNA polymerase Certain point mutations at the POL active sites of T4 and RB69 DNA polymerases, although they inactivate replication, exhibit a species-specific trans-dominant-negative phenotype, which is largely attributable to the RNA-binding translational repressor activity of the gp43 mutants This study underscores the power of the phylogenetic approach as a complement to genetics and biochemistry in analysis of structure-function relationships of proteins