1. A polyadenylylation-specific RNA-contact site on the surface of the bifunctional vaccinia virus RNA modifying protein VP39 that is distinct from the mRNA 5' end-binding "cleft".
- Author
-
Deng L, Johnson L, Neveu JM, Hardin S, Wang SM, Lane WS, and Gershon PD
- Subjects
- Base Sequence, Binding Sites, Cross-Linking Reagents, Dimerization, Methyltransferases chemistry, Methyltransferases metabolism, Models, Molecular, Mutagenesis, Site-Directed, Oligonucleotides chemistry, Oligonucleotides genetics, Polynucleotide Adenylyltransferase chemistry, Polynucleotide Adenylyltransferase metabolism, Protein Binding, Protein Conformation, RNA Caps metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Vaccinia virus genetics, Viral Proteins genetics, RNA, Messenger metabolism, RNA, Viral metabolism, Vaccinia virus metabolism, Viral Proteins chemistry, Viral Proteins metabolism
- Abstract
VP39 is a bifunctional mRNA-modifying protein that acts as both an mRNA cap-specific 2'-O-methyltransferase and a processivity factor for VP55, the vaccinia poly(A) polymerase catalytic subunit. Although regions of the protein surface required for methyltransferase function are well defined, it has been unclear whether the protein polyadenylylation function requires direct RNA contact and, if so, where the contact site(s) might be located on the protein surface. Here, we show that the VP55-VP39 heterodimer forms a stable complex with a 50mer oligonucleotide bearing a U2-N25-U motif, as opposed to the U2-N15-U motif that is optimal for stable complex formation with VP55 alone. An oligonucleotide bearing a U2-N25-U motif in which the downstream U residue is replaced with 4thioU can be efficiently photocrosslinked to VP39, but only in the context of the VP55-VP39 heterodimer. By partial proteolysis of end-labeled VP39, the site of oligonucleotide photocrosslinking was localized to the region of VP39 between residues Lys90 and Arg122. Peptide microsequencing and confirmatory mutagenesis identified the side-chain of Arg107 as the photocrosslinking site. Substitution of this residue with lysine abolished photocrosslinking entirely, consistent with the established RNA binding role of arginine in other RNA-binding proteins. This study provides clear evidence for a polyadenylylation-specific RNA-contact site on the surface of VP39, which is distinct from the RNA-binding methyltransferase "cleft" characterized in recent crystallographic and biochemical studies., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
- View/download PDF