RNA from the 5′ end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site
-
Edited by Susan R. Wessler, University of Georgia, Athens, GA, and approved August 11, 2006 (received for review June 30, 2006)
-
Fig. 1.Introduction to the R2 element. (A) R2 protein subunits bind both upstream and downstream of the 28S gene insertion site. The protein subunit bound upstream of the integration site cleaves the bottom DNA strand and uses the newly generated 3′ end to prime reverse transcription of the R2 RNA starting at the 3′ end of the R2 RNA transcript shown bound to this subunit. The protein subunit bound downstream of the integration site cleaves the top DNA strand. First (bottom) and second (top) strand cleavage sites are 2 bp apart and are each indicated with an ×. (B) Structure of the R2 ORF. The N-terminal DNA domain (gray box) has been shown to bind the downstream DNA sequences by means of highly conserved zinc-finger and myb motifs (12). The C-terminal domain (black box) encodes an endonuclease and has been proposed to bind the upstream DNA sequences. The central domain of the ORF encodes the reverse transcriptase (RT). The 5′ and 3′ RNA segments of R2 used in this study are indicated by wavy lines with numbers corresponding to the 5′ end of a full-length R2 element. (C) A specific RNA copurifies with the R2 protein. Purified R2 protein was analyzed by SDS/PAGE followed by silver staining. Lane 1 shows R2 protein after purification by our standard procedure. Lane 2 shows purified R2 protein treated with 100 ng/ml RNase A for 10 min at 37°C before loading. Lane 3 shows purified R2 protein treated with 100 μg/ml proteinase K for 10 min at 37°C before loading.
-
Fig. 2.Removal of the copurifying 5′ RNA from the R2 protein does not affect first-strand DNA cleavage (A) and TPRT activity (B) but does eliminate second-strand DNA cleavage (C). In each reaction the purified R2 protein was pretreated with RNase A (black diamond) or left untreated (gray squares). All reactions contained 10 fmol of the R2 protein and 120 fmol of the 3′ RNA, and the DNA substrate ranged from 3 to 200 fmol. At each DNA concentration, the activity of the RNase A-treated R2 protein is plotted relative to the activity conducted by the untreated R2 protein set at 1.0.
-
Fig. 3.Electrophoretic mobility shift assay (EMSA) of the R2 protein and target DNA with 1.0 pmol 3′ RNA (Left), 1.0 pmol of 5′ RNA (Center), or 0.5 pmol of both 3′ and 5′ RNA (Right). Each reaction contained 40 fmol of 32P-end-labeled target DNA and increasing amounts of RNase A-treated R2 protein. D and M refer to dimer and monomer bands as described (9).
-
Fig. 4.DNase I footprints of DNA–protein complexes containing either 3′ or 5′ RNA. (A) The target DNA was 5′-end-labeled on either the top (Left) or the bottom (Right) strand. The complexes were formed and separated on EMSA gels under conditions similar to those in lanes 2, 5, and 8 of Fig. 3. In each gel, lane 1 shows the guanosine and adenosine ladder of the DNA sequence, lane 2 shows the DNase I pattern of naked DNA, lane 3 shows the DNase I footprint of protein–DNA complexes containing the 3′ RNA, and lane 4 shows the DNase I footprint of protein–DNA complexes containing the 5′ RNA. The numbers at left correspond to phosphate positions numbered from the R2 insertion site, with positions upstream of the insertion site given negative numbers and positions downstream of the insertion site given positive numbers. (B) Summary diagram of the footprints in A. The top and bottom strand cleavage sites are marked within the DNA sequence by the vertical lines that end in triangles. Thick horizontal lines above or below the sequence represent areas of strongest protection from DNase I, thin horizontal bars represent areas more weakly protected, and vertical lines represent sites hypersensitive to DNase I.
-
Fig. 5.Effect of 5′ RNA concentration on first- and second-strand DNA cleavage. Target DNA 5′-end-labeled on either the first (A) or second (B) strand was incubated with increasing amounts of 5′ RNA. Each reaction contained 100 fmol of DNA, 12 fmol of R2 protein, and 0.43–4300 fmol of 5′ RNA. Reactions were incubated for 30 min at 37°C, and the products were separated by denaturing 6% PAGE to determine the relative levels of DNA cleavage. All values are plotted relative to the assay condition with the highest level of activity (set at 1.0).
-
Fig. 6.Factors affecting second-strand cleavage by the downstream subunit. (A) Second-strand cleavage on DNA templates that have been precleaved on the bottom strand. The precleaved substrates were made by incubation of the DNA substrate with excess R2 protein in the presence of RNase A, followed by extraction with phenol and precipitation with ethanol. Each reaction contained an excess of DNA substrate (20 fmol), and 5′ RNA (1.2 pmol) and R2 protein ranging from 1.2 to 12 fmol. Open squares indicate the fraction of the DNA bound by protein as determined on an EMSA gel, and filled squares indicate the fraction of bound DNA cleaved on the second strand as determined by 6% PAGE. (B) Effects of adding RNase A to preformed protein–5′ RNA complexes bound to DNA. The R2 protein (12 fmol) was incubated with a 5′ end-labeled top strand DNA target (33 fmol) in the presence of 1.2 pmol of 5′ RNA for 10 min at 37°C. The mixture was divided into two aliquots, with one aliquot (RNase-treated) incubated with 1 ng of RNase A for 30 min at 37°C and the other aliquot (control) mock treated. One half of each reaction was then separated on denaturing 6% polyacrylamide gels to determine the relative levels of DNA cleavage, and the second half was separated on an EMSA to determine the level of DNA binding.
-
Fig. 7.Model of R2 retrotransposition. (A) The R2 protein is composed of three domains: an N-terminal DNA-binding domain (blue shading), a central reverse transcriptase (RT) domain (green shading), and a C-terminal DNA-binding and endonuclease domain (red shading). R2 protein bound to the 3′ UTR RNA sequesters the N-terminal domain, exposing only the C-terminal DNA-binding domain for binding upstream of the insertion site (i.e., the upstream subunit conformation). Protein bound to the 5′ RNA sequesters the C-terminal DNA-binding domain, exposing only the N-terminal DNA-binding domain to bind the downstream site (the downstream subunit). (B) R2 retrotransposition is proposed to be catalyzed by two subunits in four steps. Step 1: the endonuclease (red oval) from the upstream subunit is responsible for first-strand cleavage. Step 2: the RT (green oval) of the upstream subunit catalyzes first-strand TPRT. Step 3: the downstream subunit cleaves the second DNA strand. Second-strand cleavage does not occur until reverse transcription strips away the 5′ RNA region bound to this subunit. Step 4: the downstream subunit provides the polymerase to perform second-strand TPRT. Step 4 has not yet been shown to occur in vitro.
Footnotes
- ‡To whom correspondence should be addressed. E-mail: eick{at}mail.rochester.edu
- © 2006 by The National Academy of Sciences of the USA
