Kinetics of T7 DNA polymerase conformational changes during nucleotide incorporation

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Structural and biochemical studies on the T7 DNA polymerase have revealed that a rate-limiting isomerization step preceding phosphoryl transfer contributes significantly to nucleotide selectivity. In order to directly observe enzyme conformational changes, we constructed a fluorescently labeled T7 DNA polymerase, which provides a fluorescence signal sensitive enough to report different conformational states of the enzyme. Using this conformational sensitive fluorescence (CSF), we have previously reported the equilibrium and kinetics of nucleotide-induced conformational transitions and defined distinct states for binding of correct and mismatched base pairs. In this report, we extend the study to understand the kinetics of conformation transitions during nucleotide incorporations. Our CSF data revealed that the T7 DNA polymerase cycles through open and closed states. Furthermore, kinetic studies using CSF also reveal that rates of nucleotide-induced isomerization are only slightly faster than or equal to rates of phosphoryl transfer depending on the nature of incoming substrates. The result suggests that the polymerase could utilize either isomerization or chemistry step as the kinetic barrier for nucleotide selection. In addition, kinetics of the nucleotide-induced conformational change is unchanged when normal dCTP is substituted with dCTP-alpha-S, which confirms that the thio-elemental effect originated from a reduced phosphoryl transfer rate, and we demonstrate that the chemistry step is fully rate-limiting for misincorporation. Source of research support: NIH GM071404