DNA Polymerase (Reverse Transcriptase) Specific Binding of Tryptophan Transfer RNA to Avian Myeloblastosis Virus RNA-Dependent (2006)

The ability of tryptophan tRNA (tRNATrP) to initiate reverse transcription of the of avian tumor viruses suggested that the reverse transcriptase (RNAdependent DNA polymerase; deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) might have a specific binding site for the tRNA. A complex of tRNATrP and the avian myeloblastosis virus reverse transcriptase has been demonstrated using chromatography on Sephadex G-100 columns. Of all the chicken tRNA's, only tRNATrP and a tRNA4Met bind to the enzyme with high enough affinity to be selected from a mixture of the chicken cell tRNAs. The ability of tRNATIP to change the sedimentation rate of the enzyme indicates that tRNATrP is not binding to a contaminant in the enzyme preparation. Treatment of the enzyme with monospecific antibody to reverse transcriptase prevented binding of tRNA as well as inhibited the DNA polymerase activity of the enzyme. The ability of reverse transcriptase to utilize tRNATrP as a primer for DNA synthesis, therefore, appears to involve a highly specific site on the enzyme. In vitro synthesis of DNA by the DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) found in virions of tumor viruses (reverse transcriptase) is a primer-dependent reaction (1, 2). When the 70S from virions is used as a template, a lowmolecular-weight of the complex acts as primer (3-5). For Rous sarcoma virus, this has been shown to be identical to a cellular tryptophan tRNA (tRNATrP) that is called spot 1 RNA after separation by 2-dimensional gel electrophoresis (6-9). Such tRNATrP has been isolated from three sources: RNA, 4S from virions of Rous sarcoma virus, and normal chicken embryo fibroblasts (4, 5, 8). Although other tRNAs are found associated with and as 4S in virions (10), 90% of the DNA molecules copied from have been shown to be initiated using tRNATrP as a primer (4). Part of its ability to act as primer may result from the tight association of tRNATrP with the 35S subunits of the RNA; of the 70S-associated tRNAs, tRNATrP requires the highest temperature for removal from the 35S (4, 11). The tRNATrP also appears to be able to anneal back to 35S (12, 13). The association of tRNATrP with 35S RNA, however, is not sufficient to explain its ability to act as primer for the reverse transcriptase because, in general, the enzyme uses DNA efficiently as primer but utilizes primers poorly (1, 14). The apparent selectivity of reverse transcriptase for tRNATrP over other 70S-associated tRNAs and the poor rate of priming of reverse transcriptase by homopolymers led us to investigate whether there might be a specific binding site on the reverse transcriptase for tRNATrP. Abbreviation: AMV, avian myeloblastosis virus., 2535 Using an assay for high-affinity binding sites, we have been able to demonstrate specific binding of tRNATrP to the reverse transcriptase of avian myeloblastosis virus (AMV). MATERIALS AND METHODS Materials. AMV reverse transcriptase was purified by sequential chromatography on columns of DEAE-Sephadex and phosphocellulose (15). The phosphocellulose fraction was further purified on a DNA-cellulose column (16). The enzyme preparation gave only two bands after electrophoretic separation on 10% sodium dodecyl sulfate-polyacrylamide gels; these migrated as do the previously characterized a and ,B subunits of AMV reverse transcriptase (17). Antiserum to AMV reverse transcriptase was obtained from Dr. R. Nowinski, Madison, Wisc. (18). The IgG fraction was purified from serum as described (19). Chicken liver tRNA and chicken liver aminoacyl tRNA synthetases were prepared following published procedures (20-22). Purified Escherichia coli tRNATyr, E. coli tRNASer, and yeast tRNAPhe were gifts from Dr. U. L. RajBhandary and M. Silberklong. (dG)12 18 and (dT)12-18 were purchases from Collaborative Research Corp. These polynucleotides were labeled at their 5'-end using polynucleotide kinase and ['y-32P]ATP (23). Radioactive nucleotides and amino acids were products of New England Nuclear Corp. Sephadex G-100 was obtained from Pharmacia Corp. Preparation of 32P-Labeled RNAs. Cultures of chicken embryo fibroblasts infected with Schmidt-Ruppin D Rous sarcoma virus or Carr-Zilber associated virus were obtained from Dr. H. M. Temin. The cells were radioactively labeled in 100 mm diameter dishes with [32P]orthophosphate as described previously (7), using 1-2 mCi of [32P]orthophosphate per ml of medium. After 48 hr of labeling, virus was harvested from the medium by centrifugation and was extracted without further purification (7). Viral was fractionated into and free 4S by centrifugation through sucrose gradients (7). The 4S was precipitated with 2 volumes of ethanol, resuspended in 0.05 M NaCl, 0.01 M Tris-HCI, pH 7.8, 0.01 M MgCl2, and was applied to a 1 ml column of DEAE-Sephadex A-50 (Pharmacia). After successive washes with 0.05 M NaCl and 0.3 M NaCl the was eluted from the column with 1 M NaCl in the same buffer and concentrated by ethanol precipitation. After virus was harvested, the labeled cells were washed twice with 5 ml of TSE buffer (0.02 M Tris-HCI, pH 7.8; 0.1 M NaCl; 1 mM EDTA) at room temperature. Cells were then treated on the culture plates with 0.5% sodium dodecyl sulfate in TSE buffer, 1 ml/10 cm plate for 15 min at room temperature. The suspension was carefully removed from 2536 Biochemistry: Panet et al. the plates to avoid shearing of DNA and 0.25 volume of 5 M NaCl was added. After storage overnight, the precipitate was removed by centrifugation at 15,000 X g for 20 min. The supernatant was extracted twice with phenol and twice with chloroform/isoamyl alcohol (24:1). Nucleic acids were precipitated with 2 volumes of ethanol at -20° in the presence of 100 ,g of carrier (phenol-extracted Sigma type IV yeast RNA). was purified by chromatography on DEAE-Sephadex as described above, followed by centrifugation in a 5-20% sucrose gradient in TSE buffer containing 0.5% Sarkosyl (Geigy) at 49,000 rpm, 2 hr, 40 in a Beckman SW 50.1 rotor. The. individual tRNA species were purified from virionassociated or cellular tRNA using 2-dimensional gel electrophoresis as described previously (7). Unlabeled tRNATrP from chicken liver was purified as described elsewhere (9). E. coli 32P-labeled 5S and 4.5S were prepared as described previously (6) using the hot sodium dodecyl sulfate method of Bremer and Yuan (24). Binding Assay. For assay of binding, the reaction mixture (100-200 ,l) contained 50 mM Tris-HCl (pH 8.3), 60 mM NaCl, 10 mM 2-mercaptoethanol, 10 mM MgCl2, 5% (v/v) glycerol, 0.1% Nonidet P-40, [32P]RNA as indicated, and 50 to 150 enzyme units of reverse transcriptase. After 10 min incubation at 20, the reaction mixtures were chromatographed on columns of Sephadex G-100 (0.6 X 23 cm) preequilibrated at 40 with buffer A (0.1 M potassium phosphate, pH 7.5, 5 mM MgC12, 10 mM 2-mercaptoethanol, 10% (v/v) glycerol, 0.1 mM EDTA, and 0.1% Nonidet P-40). The column was eluted with buffer A. Fractions of 0.17 ml were collected and 25-50 ,ul portions of each fraction were assayed for reverse transcriptase activity; 60-70% of added enzymatic activity was routinely recovered in the fractions. Portions of each of the fractions were also used for determination of [32P]RNA. Enzyme Assay. Reaction mixtures (100 Al) contained 50 mM Tris-HCl (pH 8.3), 6 mM MgCl2, 0.1 mM [3H]dGTP (100 cpm/pmol), 2 Mg of poly(C), 0.5 Mig of (dG)1218, 5 mM dithiothreitol, and 25-50 Ml of fractions from the Sephadex G-100 columns or glycerol gradients. Reactions were incubated for 30 min at 37'. One enzyme unit was defined as the amount needed to catalyze the incorporation of 1 pmol of dGMP into polymer in 1 min. The assay for E. coli DNA polymerase I was as described for reverse transcriptase but 1 Mg of poly(dA) and 1 Mg of (dT)12_18 were used as the template and primer and the incorporation from [3H]dTTP (60 cpm/pmol) was followed.