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Interplay of two uridylate‐specific RNA binding sites in the translocation of poly(A) polymerase from vaccinia virus
Author(s) -
Deng Li,
Gershon Paul D.
Publication year - 1997
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/16.5.1103
Subject(s) - vaccinia , rna processing , polymerase , biology , virus , rna , microbiology and biotechnology , virology , biochemistry , dna , gene , recombinant dna
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, rU 34 , in VP55 binding and salt‐resistant polyadenylylation. The positions of the two uridylate interaction sites, ∼10 and ∼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 ∼10 nt further upstream.