RNA binding characteristics and overall topology of the vaccinia poly(A) polymerase-processivity factor-primer complex.
The vaccinia virus-encoded heterodimer responsible for poly(A) tail elongation comprises a polyadenylylation catalytic subunit (VP55) and associated processivity factor (VP39). We show that monomeric VP39's affinity for RNA homopolymers follows the hierarchy poly(I) >poly(U) >>poly(G) >poly(A) >poly(C), that the heterodimer interacts stably with 40-45 nucleotide nucleic acid segments, and that its homopolymer preference for polyadenylylation priming is comparable to the VP39 affinity hierarchy (above). For oligonucleotide ligands possessing the previously-identified (rU)2-(N)25-rU heterodimer-binding motif, the heterodimer's affinity and base-type preference are mediated via both the (rU)2and rU portions, with the greater contribution coming from (rU)2. VP39's R107 sidechain contributes to specificity at the downstream rU. Substitution of each ribouridylate of the motif with either ribothymidine or 4-thiodeoxythymidine indicated that the downstream rU interacts with both heterodimer subunits, whereas the upstream (rU)2interacts only with VP55. A 'crosslinking SELEX' approach indicated VP39-base proximity around position -10 of a 4-thioribouridine/deoxycytidine ligand pool. Upon incubating the heterodimer with a panel of identical-sequence oligonucleotides derivatized with azidophenacyl bromide at various phosphate positions, those derivatized at positions -11 to -21 photocrosslinked to both subunits in a coordinated manner. This region may therefore pass through a 'cleft' or enclosed 'channel' at the subunit interface.
Johnson
L
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Department of Medical Biochemistry and Genetics/Institute of Biosciences and Technology, Texas A&M University, Houston, TX 77030, USA.
Gershon
P D
PD
D001665:Binding Sites; D009690:Nucleic Acid Conformation; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D014616:Vaccinia virus; D014764:Viral Proteins / Q000378:metabolism*
D012367:RNA, Viral; C088682:VP39 protein, Vaccinia virus; D014764:Viral Proteins; D012313:RNA; D011062:Polynucleotide Adenylyltransferase