The poly(A) tail is a virtually ubiquitous mRNA feature, yet neither its role(s) nor its mechanism(s) of synthesis are well characterized in any organism. Of the various tail-synthesizing enzymes (poly(A) polymerases of PAPs), the vaccinia virus enzyme provides a powerful tool with which to study important aspects of PAP mechanism. Thus, although it shares characteristics with the cellular PAPs. the viral enzyme participates in a relatively simple polyadenylylation process that is coupled to neither the interactions or auxiliary protein factors with multiple RNA signals, nor other cellular processes such as endonucleolytic cleavage, mRNA splicing or protein phosphorylation. I propose to capitalize upon the viral enzyme's directness of action to investigate mechanisms central the action of any PAP, namely catalysis, translocation and processivity. Mechanistic insights regarding PAP, of one of the simplest known RNA- synthetic enzymes, should contribute to a more general understanding of RNA synthesis and macromolecular machinery. The VP55 (catalytic) subunit of the heterodimeric vaccinia PAP abruptly ceases processive primer elongation after producing tails approximately 25-30 nt in length, and the VP39 subunit is a processivity factor for further elongation by VP55, the heterodimer and the heterodimer-RNA ternary complex will be investigated using various complementary techniques (such as site- specific photocrosslinking, crosslinking-SELEX, photocrosslinking with label-transfer and an anchored 'chemical protease' approach) to identify topological constraints. For example, one aim is to establish whether VP39's polyadenylylation-specific RNA contact site(s) like within a 'fully' on the protein surface between two parts of an apparently bipartite dimerization interface. Initial attempts to improve VP55 over- expression should facilitate elucidation of its 3D structure. RNA determinants of VP55 translocation will be investigated using both discrete oligonucleotides and a novel selection scheme. The relationship between VP55 translocation, primer anchoring and processive tail synthesis will be established using covalently-photocross-linked PAP- primer complexes and flexibility in the relative positioning of the 3' OH-distal and proximal primer-PAP contact sites will be characterized using oligonucleotides designed to 'squeeze' and 'stretch' their relative positions. Finally, to investigate roles for metal ions at VP55's catalytic center, we will determine which of the two apparent divalent cation binding sites in the VP55.ATP complex is Mn2+-enhanced; identify roles for divalent metals in NTP binding and catalysis using co-valently photocrosslinked VP55-primer conjugates; quantify VP55-metal ion affinities by EPR; and address metal ion coordination using unusual, sulfur-substituted oligonucleotides.
Showing the most recent 10 out of 19 publications
