A. Poly(A) Tail Addition
The vaccinia PAP is unusual in being able to rapidly and processively add long poly(A) tails to RNA substrates without requiring the presence of specific RNA sequences. Conditions for doing this vary, depending upon the importance of complete substrate utilization and regulated tail length. Reaction variables include
(1) whether the VP55 subunit alone or both subunits are present in the reaction;
(2) the time and temperature of the reaction; (3) whether Mn2+ is present as well as/in place of Mg2+; (4) concentrations of monovalent cation; (5) relative concentrations of PAP, RNA, and ATP; and (6) the presence/absence of ATP
analogs. Instead of exhaustively discussing these parameters, some appropriate PAP assays will be described and pointers provided to the primary literature.
An electrophoretic time-course PAP assay has been described and subsequently refined (Gershon and Moss, 1992, 1993a,b; Shuman and Moss, 1988). This assay employs a discrete 5' end-labeled RNA primer, and polyadenylylation products are analyzed by gel electrophoresis. As originally reported (Shuman and Moss, 1988), reaction conditions were similar to those described for [a-32P]AMP incorporation into trichloroacetic acid (TCA)-precipitable RNA (Gershon and Moss, 1996; Moss et ai, 1975), except for a reduction of the MnCl2 concentration to 0.6 mM, the omission oflabeled ATP, and the use ofa small (10-//.1) reaction volume and the RNA primer Ui0 after 5 '-end labeling of the latter using [y-32P] ATP and T4 polynucleotide kinase. After a "time zero" sample is removed, reactions are initiated by adding enzyme to a mixture ofthe other components. Aliquots of the reaction mixture are then withdrawn at various times and mixed with equal volumes of deionized formamide. Reaction products are analyzed by electrophoresis in polyacrylamide gels containing 7 Ai urea and Tris-borate-EDTA (TBE) buffer followed by autoradiography. Modifications have been made to this assay (Gershon and Moss, 1992, 1993a,b). Specifically, chemically or enzymatically synthesized primers have been used, which are generally more than 25 nt in length. These are normally gel-purified after synthesis but prior to labeling. Labeling is usually terminated by incubating labeling reactions at 65°C for 15 min, obviating a second gel-purification step. In general, primer is used in more than threefold molar excess over enzyme. For the majority of polyadenylylation reactions, MnCl2 is replaced by MgCl2. In reactions where MnCl2 is still used, MnCl2 • 4H20 is dissolved in water immediately prior to use, and the MgCl2, Tris-HCl, and DTT in the assay mixtures, and in the polynucleotide kinase labeling reactions, are substituted for by MnCl2, N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid-sodium hydroxide (HEPES-NaOH), and 2-mercaptoethanol, respectively (Thomson and Gershon, 1995). The above polyadenylylation assay is effective when used with either monomelic VP55, the reconstituted PAP subunits, or the purified heterodimer.
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