Interplay of two uridylate-specific RNA binding sites in the translocation of poly(A) polymerase from vaccinia virus

被引:17
作者
Li, DN [1 ]
Gershon, PD [1 ]
机构
[1] TEXAS A&M UNIV,DEPT BIOCHEM & BIOPHYS,INST BIOSCI & TECHNOL,HOUSTON,TX 77030
关键词
poly(A); polymerase; RNA; selection; translocation; STRUCTURAL-ANALYSIS; CATALYTIC SUBUNIT; TERNARY COMPLEXES; POLYADENYLATION; ELONGATION; CLEAVAGE; TAIL;
D O I
10.1093/emboj/16.5.1103
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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, similar to 10 and similar to 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 similar to 10 nt further upstream.
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页码:1103 / 1113
页数:11
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