Article Figures & Data
Figures
- FIG. 1.
R2 elements and the TPRT mechanism. (A) Diagram of the R2 TPRT mechanism. RNA corresponding to the 3′-UTR RNA of the R2 element and the R2 protein form a specific RNP complex. The RNP complex binds the target DNA and cleaves the first (bottom) strand. The 3′ DNA end generated by this nick is used to prime first-strand cDNA. Second (top)-strand cleavage occurs after reverse transcription. (B) R2 elements insert into a specific site within the 28S rRNA gene. R2 elements encode a 120-kDa protein containing an N-terminal domain with cysteine-histidine zinc fingers and c-Myb DNA binding motifs, a central reverse transcriptase domain (RT), and a C-terminal domain with a restriction-like-endonuclease domain and putative nucleic acid binding motif(s). Indicated below the R2 diagram is the 3′-UTR sequence used as R2 RNA in this study and the N-terminal peptide used for Fig. 3B and C.
- FIG. 2.
EMSA of the R2 protein and target DNA in the presence and absence of RNA. The triangles above the lanes represent the relative protein concentrations between the various lanes (12 to 360 fmol/reaction). The DNA and RNA substrates used for a given reaction are indicated above each lane. An asterisk indicates that the substrate was 32P labeled. The R2 protein was allowed to bind to substrate DNA at 37°C for 15 min (lanes 4 to 13) or 30 min (lanes 1 to 3). DNA cleavage was prevented by the presence of EDTA (lanes 1 to 3) or by using an endonuclease-mutant R2 protein (lanes 4 to 13). Shifted complexes, a, b, c, and d, in the remainder of this report are referred to as follows: M−, protein monomer without RNA; M+, monomer with RNA; D−, protein dimer without RNA; and D+, dimer with RNA. The structures of each complex based on the footprint studies shown in Fig. 3 are diagramed to the left of the gel.
- FIG. 3.
Bipartite binding of the R2 protein revealed by DNase I footprints. (A) Comparison of the DNase I footprints for the monomer (M+, lane 3) and the dimer (D+, lane 2). The target DNA was 5′end labeled on either the top (left panel) or bottom strand (right panel). The endonuclease mutant was used in order to observe the complexes prior to cleavage. Lane 1, DNase I pattern of naked DNA; lanes labeled G+A, guanosine-plus-adenine ladders. Numbers to the left of the footprint correspond to base positions relative to the R2 cleavage dyad (see panel D). (B) Electrophoresis mobility shift assay in the presence (+) and absence (−) of a 140-amino-acid N-terminal peptide of the R2 protein containing the zinc-finger and c-Myb motifs. Free DNA and bound DNA are labeled 1 and 2, respectively. (C) DNase I footprint of the shifted N-terminal peptide bound to DNA (lane 2) compared to free DNA (lane 1) in each panel. (D) Summary of the R2 protein footprints. The 2-bp staggered cleavage by R2 is indicated by the lines ending in triangles. Nucleotide positions are numbered relative to the dyad cleavage site, with negative numbers corresponding to upstream sequences (relative to transcription of the 28S gene; see Fig. 1B). Thick horizontal lines represent areas of greatest protection by the R2 protein, while thinner horizontal lines indicate weaker protection. Thick vertical lines are DNase I-hypersensitive sites.
- FIG. 4.
Endonuclease activities associated with the M+ and D+ complexes. (A) Time course of R2 binding and cleavage as revealed by EMSA. The 184-bp DNA substrate was 5′ end labeled on either the top (lanes 1 to 5) or bottom strand (lanes 6 to 8). The individual time points were 1, 3, 12, 20, and 30 min at 37°C; the 20-min time point was omitted in the bottom-strand assay. The M+ and D+ complexes are diagramed as in Fig. 2 except that DNA cleavage is indicated by a gap in the DNA. After top-strand cleavage, the R2 protein remains bound to the upstream DNA (ΔM+) but releases the downstream DNA (ΔDNA). (B) The cleavage state of the 184-bp DNA in the various complexes shown in panel A excised and run on a denaturing gel. Equivalent amounts of radioactive counts were loaded onto each lane of the gel. (C) Effects of 3′-UTR RNA concentration on DNA cleavage. Assays were conducted with the target DNA 5′ end labeled on either the top or bottom strand. Each reaction had 12 fmol R2 protein, 20 fmol 184-bp 5′ end-labeled DNA, 8 ng poly(dIdC), and 0.124 to 12400 fmol 3′-UTR RNA. Half of each assay was analyzed by EMSA to determine the fraction of DNA bound by the protein, and the second half was analyzed by denaturing gel electrophoresis to determine the fraction of the DNA cleaved. The EMSA gel (top panel) is from the bottom strand-labeled assay, and the graph (bottom panel) is from the top strand-labeled assay. It should be noted that the M− complex contained two conformations, a predominant slower-migrating form in which the target DNA was cleaved on the bottom strand and a faster-migrating noncleaved form (see reference 9 and Fig. 3 and 7).
- FIG. 5.
Reverse transcriptase activities associated with the M+ and D+ complexes. (A) Effect of protein concentration on the TPRT reaction. The y axis represents the fraction of the total DNA that was bound by protein, cleaved on the top or bottom strand, or had undergone TPRT. A value of 1 indicates 100% of the DNA in the reaction. The x axis is a logarithmic scale of protein concentration (fmol in each reaction). Each reaction was a standard TPRT reaction (see Material and Methods) with the 184-bp DNA substrate, the 250-nt R2 RNA, and R2 protein in the range of 0.12 to 360 fmol. The data are derived from two separate reactions (bottom or top strand end labeled) with reaction products analyzed on both EMSA and denaturing polyacrylamide gels. In the labeled bottom-strand reaction, a ∼340-nt product represented the DNA that had undergone TPRT, while the combined DNA corresponding to the ∼340-nt product and the 93-bp product represented the total level of bottom-strand cleavage. The level of top-strand cleavage was determined in the labeled top-strand reaction by the accumulation of an 89-nt product. (B) Effect of protein concentration on TPRT and top-strand cleavage. The y axis is the fraction of DNA that had undergone TPRT (triangles) or top-strand (circles) cleavage divided by the amount of DNA bound at each protein concentration. The x axis is the same as in panel A. The data presented are the combined results of two independent experiments. (C) EMSA analysis of a TPRT reaction at a low protein concentration. Reactions were preincubated for 30 min (lane 1) to reduce the presence of dimers, then 25 μM dNTPs were added and the incubation continued for 15 min (lane 2). Reactions were standard TPRT assays except that the amount of protein was only 4 fmol. Complexes that have undergone TPRT are marked (M+TPRT). R−TPRT is an artifact resulting from denaturation of the bottom 17-bp downstream DNA after TPRT. (D) Levels of TPRT and top-strand cleavage in the complexes in panel C. Lanes 1 to 3, complexes excised from the EMSA gel and the DNA analyzed on a denaturing polyacrylamide gel. Lane 4, a separate reaction similar to that in panel C but with top strand labeled to monitor top-strand cleavage.
- FIG. 6.
Sequential addition of protein subunits to the DNA target site. (A) TPRT assays. Near-saturating levels (140 fmol) of wild-type (RT+) or reverse transcriptase-deficient protein (RT−) were added to the reaction and allowed to bind the bottom strand-labeled DNA substrate (60 fmol) for 17 min at 37°C. The reaction was split in two, and 5× more protein (350 fmol), either RT− or RT+, was added along with 25 μM dNTPs (final concentration) for an additional incubation at 37°C for 17 min. The bar graph reports the relative level of TPRT attained in each reaction. (B) Second-strand cleavage. Similar to panel A, wild-type R2 protein (EN+) was added to the reaction and allowed to bind the top strand-labeled DNA substrate. The reaction mixture was split in two, and 5× more protein, either EN+ or EN−, was added before the second incubation.
- FIG.7.
Model of a complete R2 integration reaction. (A) The R2 ORF is divided into three domains: N-terminal DNA binding (blue), reverse transcriptase (green), and C terminal (red) (5). The C-terminal domain has been subdivided into DNA-binding and endonuclease subdomains. The DNA cleavage domain (red oval with spike) is on a flexible linker, similar to the type IIs restriction endonucleases. In the context of a dimer, each R2 subunit exposes only one DNA binding domain, while the other domain is sequestered. The subunit with bound 3′-UTR RNA exposes the C-terminal DNA-binding domain and binds upstream of the cleavage/integration site. The subunit lacking RNA exposes the N-terminal DNA binding domain and binds in opposite orientation downstream of the site. While it is likely that protein-protein interactions play a role in dimer formation, such interactions have not been depicted. Step 1 (first-strand cleavage). The EN domain of the upstream subunit cleaves the bottom strand. Step 2 (first-strand synthesis). The upstream subunit undergoes a conformational change, placing its reverse transcriptase over the bottom strand cut, which can then catalyze TPRT. Step 3 (second-strand cleavage). The EN domain of the downstream subunit cleaves the top strand. Step 4 (second-strand synthesis). The upstream subunit undergoes a conformational change, placing its RT over the top-strand cut, which can then catalyze second-strand synthesis. This last step does not occur in our in vitro reaction. (B) Model of L1 integration. L1 elements encode two ORFs (27). The second ORF contains a N-terminal AP endonuclease domain (red), a reverse transcriptase domain (green), and a C-terminal domain, which is postulated in the model to be DNA binding (blue). As in the R2 model, the active complex is assumed to be a dimer, with each subunit in opposite orientation conducting one-half of the reactions. One subunit binds RNA, binds the DNA target by means of the APE domain, cleaves the first strand, and conducts TPRT. The second subunit binds by means of the C-terminal domain, cleaves the second strand, and conducts second-strand DNA synthesis. No evidence yet exists for the L1 C-terminal domain binding DNA.
