Use of vaccinia virus poly(A) polymerase for RNA 3'-end labeling with a chain-terminating nucleotide or a short 3' homopolymer tract.

Abstract

Conditions are described for the 3'-end labeling of RNA with 32P 3'-dATP (3'-deoxyadenosine-5'-triphosphate), a chain-terminating nucleotide, using the poly(A) polymerase (PAP) encoded by vaccinia virus. Reaction time, divalent cation species and concentration, and the requirement for both subunits of the PAP were investigated. In the presence of Mn2+, vaccinia PAP is able to tail RNA primers with tracts of 3'-oligo(U), oligo(C) and oligo(G). Conditions for the addition of labeled 3'-homopolymer tracts were characterized. The use of low nucleotide concentrations in this study revealed an apparently fixed divalent cation concentration optimum of 0.1 mM, distinct from the previously noted requirement for a 1:1 divalent cation:NTP complex. This indicates a possible requirement for multiple divalent cations in nucleotidyl transfer by vaccinia PAP.

• Authors: J G Thomson, P D Gershon
• Journal: BioTechniques (Biotechniques) Vol. 19 Issue 3 Pg. 416-20, 422-5 (Sep 1995) ISSN: 0736-6205 [Print] England
• PMID: 7495555 (Publication Type: Research Support, Non-U.S. Gov't, Technical Report)

Chemical References

• Cations, Divalent
• Deoxyadenine Nucleotides
• Deoxyadenosines
• Phosphorus Radioisotopes
• Manganese
• RNA
• Polynucleotide Adenylyltransferase
• cordycepin
• Magnesium
• 2'-deoxyadenosine triphosphate

Topics

• Cations, Divalent
• Deoxyadenine Nucleotides (metabolism)
• Deoxyadenosines
• Gene Expression
• Kinetics
• Magnesium (pharmacology)
• Manganese (pharmacology)
• Phosphorus Radioisotopes
• Polynucleotide Adenylyltransferase (genetics, metabolism)
• RNA (metabolism)
• Vaccinia virus (enzymology, genetics)