Molecular flexibility and discontinuous translocation of a non-templated polymerase.
Little is known regarding the translocation of non-templated nucleic acid polymerases with respect to single-stranded primers. VP55, the vaccinia virus poly(A) polymerase, translocates as it processively adds a approximately 3-7 adenylate tail to primers possessing only three ribouridylate residues (as an (rU)(2)-N(15)-rU motif), and a approximately 25-30 adenylate tail to primers that are more U-rich. Here, three models were addressed for the translocation of VP55 with respect to its primer, namely: (a) rigid protein/rigid nucleic acid; (b) flexible protein/rigid nucleic acid; (c) rigid protein/flexible nucleic acid. Analysis of free and covalently VP55-attached primers favored either (b) or a version of (c) incorporating a passive steric block, and suggested two regions of relative motion between polymerase and primer. Inclusion of a 6nt uridylate-rich patch at the primer 3' end switched the polymerase from approximately 3-7 nt to approximately 25-30 nt tail addition without affecting initial binding affinity. By synthesizing this patch as a (rU/dC) pool, discontinuous polymerase movements could be detected.
Johnson
L
L
Department of Medical Biochemistry and Genetics, Institute of Biosciences and Technology, Texas A and M University System Health Science Center, Houston, TX 77030, USA.
Liu
S
S
Gershon
P D
PD
D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D012313:RNA / Q000096:biosynthesis*; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
D012313:RNA; D011062:Polynucleotide Adenylyltransferase