Saltatory forward movement of a poly(A) polymerase during poly(A) tail addition

Vaccinia poly(A) polymerase (VP55) interacts with > or = 33-nucleotide (nt) primers via uridylates at two sites (-27/-26 and -10). It adds approximately 30-nt poly(A) tails with a rapid, processive burst in which the first few nt are added without substantial primer movement, and addition of the remaining adenylates is dependent upon a six-uridylate tract at the extreme 3' end of the primer and accompanied by polymerase translocation. Interaction of VP55 with 2-aminopurine (2-AP)-containing primers was associated with a 3-fold enhancement in 2-AP fluorescence. In stopped-flow experiments, fluorescence intensity changed with time during the polyadenylation burst in a manner dependent upon the position of 2-AP, indicating a non-uniform isomerization of the polymerase-primer complex with time consistent with a discontinuous (saltatory) translocation mechanism. Three distinct translocatory phases could be discerned: a -10(U)-binding site forward movement, a -27/-26(UU)-binding site jump to -10, then a -27/-26(UU)-binding site movement further downstream. Poly(A) tail elongation showed no apparent pauses during these isomerizations. Fluorescence changes during polyadenylation of 2-AP-containing primers with short preformed oligo(A) tails reinforced the above observations. Primers composed entirely of oligo(U) (apart from the 2-AP sensor), in which the polymerase modules might be most able to "slide" uniformly, also showed the characteristic saltatory pattern of translocation. These data indicate, for the first time, a discontinuous mode of translocation for a non-templated polymerase.