Interplay of two uridylate-specific RNA binding sites in the translocation of poly(A) polymerase from vaccinia virus.
The VP55 (catalytic) subunit of vaccinia virus heterodimeric poly(A) polymerase (PAP) contacts 31-40 nucleotide segments of RNA in a uridylate-dependent manner, and effects the rapid, processive addition of a 30 nt oligo(A) tail. Here, the minimum size of uridylate-containing RNA required for stable VP55 interaction was refined to 33-34 nt. VP55 binding experiments using a set of sixteen 34 nt DNA-RNA chimeras, each containing a differently positioned tetra-uridylate cluster within an oligo(dC) background, indicated that the protein contacts uridylates at two positions within the oligonucleotide. Combination of two optimally positioned tetra-uridylate clusters into a single oligonucleotide fully restored the properties of an optimal substrate, rU34, in VP55 binding and salt-resistant polyadenylylation. The positions of the two uridylate interaction sites, approximately 10 and approximately 25 nt from the oligonucleotide 3' OH, were confirmed using a selection scheme employing dC-rU oligonucleotide chimera pools. These and additional data suggest a mechanism for polymerase translocation with respect to RNA comparable with inchworming models of transcriptional elongation. In selection experiments incorporating the PAP-associated processivity factor VP39, the latter was shown to replace the 3' OH-distal uridylate contact site with one approximately 10 nt further upstream.
Deng
L
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Department of Biochemistry and Biophysics/Institute of Biosciences and Technology, Texas A&M University, Houston 77030-3303, USA.
Gershon
P D
PD
D001665:Binding Sites; D006868:Hydrolysis; D008024:Ligands; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D014542:Uridine Monophosphate / Q000378:metabolism*; D014616:Vaccinia virus / Q000201:enzymology* / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
D008024:Ligands; D009843:Oligoribonucleotides; C088682:VP39 protein, Vaccinia virus; D014764:Viral Proteins; D012313:RNA; D014542:Uridine Monophosphate; D011062:Polynucleotide Adenylyltransferase