Abstract
Reverse transcriptase-mediated RNA displacement synthesis is required for DNA polymerization through the base-paired stem portions of secondary structures present in retroviral genomes. These regions of RNA duplex often possess single unpaired nucleotides, or "bulges," that disrupt contiguous base pairing. By using well defined secondary structures from the human immunodeficiency virus, type 1 (HIV-1), genome, we demonstrate that removal of these bulges either by deletion or by introducing a complementary base on the opposing strand results in increased pausing at specific positions within the RNA duplex. We also show that the HIV-1 nucleocapsid protein can increase synthesis through the pause sites but not as efficiently as when a bulge residue is present. Finally, we demonstrate that removing a bulge increases the proportion of strand transfer events to an acceptor template that occur prior to complete replication of a donor template secondary structure. Together our data suggest a role for bulge nucleotides in enhancing synthesis through stable secondary structures and reducing strand transfer.
Highlights
Two strand transfer reactions are required to complete reverse transcription of the retroviral genomic RNA
HIV-1 Reverse transcriptase (RT) was added to extension reactions, and accumulation of the full-length extension products was monitored over time
We have demonstrated that removing single nucleotide bulges in the stem of either the TAR element or poly(A) signal, either by deleting nucleotides or inserting matching nucleotides, results in increased pausing within the duplex portion of the hairpin (Figs. 1, 2, and 5)
Summary
Materials—Reagents were obtained as follows: HIV-1 reverse transcriptase from Worthington; restriction enzymes, T4 polynucleotide kinase, T4 DNA polymerase, T4 DNA ligase, and Vent polymerase for PCR from New England Biolabs; Plasmid pGEM9Zf(Ϫ) and the T7 and SP6 RiboMax transcription kits from Promega; [␥-32P]ATP from PerkinElmer Life Sciences; Pfu DNA polymerase for site-directed mutagenesis from Stratagene; and reagents used for RNA solutions from Ambion. Primer extensions were started by the addition of dNTPs. Final concentrations in a total volume of 20 l were as follows: 50 mM Tris, pH 8.0, 50 mM KCl, 10 mM DTT, 5 mM MgCl2, 100 M of each dNTP, 50 nM primer, 100 nM template, and 700 nM RT. MgCl2, DTT, and 2.5 pmol of HIV-1 RT (final concentration 250 nM) were added except in the case of the pre-trap in which a mixture of 200 g of heparin and 30 pmol of an unlabeled DNA/RNA primer/template substrate was added first. The primer/donor template ratio was 1:1 (final concentration 50 nM each), and after addition of the DTT, MgCl2, and 7 pmol of HIV-1 RT and incubation for 5 min at 37 °C, 37.5 pmol of HIV-1 NC was added, and the reaction was incubated for an additional 5 min. The final ratio of donor to acceptor RNA template was 1:3
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