Although the poly (A) tail is a nearly ubiquitous feature of mRNA, the structure and dynamics of the enzyme that adds it are largely a black box. The vaccinia virus poly (A) polymerase (PAP) appears to be unique among the non-templated nucleic acid polymerases in possessing (a) an innate ability to translocate, (b) a processivity factor that can assist the polymerase in elongation. Moreover, the vaccinia enzyme is unusual in adding a tail in the absence of coupled processes such as RNA cleavage signal recognition, RNA endonucleolytic scission, mRNA splicing and protein phosphorylation. The vaccinia virus PAP therefore provides a powerful tool with which to study important aspects of polymerase molecular dynamics within a single-chain enzyme. Although the isolated catalytic (VP55) subunit of the vaccinia PAP heterodimer can processively elongate a primer, polyadenylation halts after tails approximately 25 - 30 nt in length have been added. The VP39 subunit is a processivity factor for tail elongation by VP55. VP39's structure and properties were studied during the initial funding period. The second funding period continued with a characterization of the translocational properties of the VP55 subunit, and the topography of both the heterodimer and the heterodimer-RNA ternary complex. Very recently, a long-standing obstacle to the study of VP55, namely its high-level expression was overcome, opening the door to structural and functional approaches requiring larger amounts of VP55 protein.
Aim I of this competing continuation addresses the structural biology and structure-function relationships within the polymerase-processivity factor (VP55-VP39) heterodimer.
Aims 2 and 3 address polymerase molecular dynamics. It is proposed that techniques in use in the P.l.'s lab (molecular biological approaches, photo crosslinking, mass spectrometry) will be augmented by inter-lab collaboration for the application of stopped-flow fluorescence and atomic force microscopy. It is expected that the proposed approaches will provide an integrated view of the structure and dynamics of a translocating, non-templated polymerase during formation of the poly (A) tail.
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