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. 2008 May 9;283(19):12756-62.
doi: 10.1074/jbc.M705003200. Epub 2008 Feb 20.

G4-forming sequences in the non-transcribed DNA strand pose blocks to T7 RNA polymerase and mammalian RNA polymerase II

Silvia Tornaletti  1 , Shaun Park-Snyder, Philip C Hanawalt
Affiliations

G4-forming sequences in the non-transcribed DNA strand pose blocks to T7 RNA polymerase and mammalian RNA polymerase II

Silvia Tornaletti et al. J Biol Chem. .

Abstract

DNA sequences rich in runs of guanine have the potential to form G4 DNA, a four-stranded non-canonical DNA structure stabilized by formation and stacking of G quartets, planar arrays of four hydrogen-bonded guanines. It was reported recently that G4 DNA can be generated in Escherichia coli during transcription of plasmids containing G-rich sequences in the non-transcribed strand. In addition, a stable RNA/DNA hybrid is formed with the transcribed strand. These novel structures, termed G loops, are suppressed in recQ(+) strains, suggesting that their persistence may generate genomic instability and that the RecQ helicase may be involved in their dissolution. However, little is known about how such non-canonical DNA structures are processed when encountered by an elongating polymerase. To assess whether G4-forming sequences interfere with transcription, we studied their effect on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We used a reconstituted transcription system in vitro with purified polymerase and initiation factors and with substrates containing G-rich sequences in either the transcribed or non-transcribed strand downstream of the T7 promoter or the adenovirus major late promoter. We report that G-rich sequences located in the transcribed strand do not affect transcription by either polymerase, but when the sequences are located in the non-transcribed strand, they partially arrest both polymerases. The efficiency of arrest increases with negative supercoiling and also with multiple rounds of transcription compared with single events.

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Figures

FIGURE 1.
FIGURE 1.
A, DNA substrate used in this study. DNA template pUC15RNTS for T7 and RNAP II transcription consisted of closed circular DNA molecules containing the Ig Sμ repeat cloned downstream of the T7 or adenovirus major late promoter (AdLMP), constructed as described under “Experimental Procedures.” The transcription start sites (+1) are represented by bent arrows. B, T7 RNAP transcription of substrates containing G-rich sequences in the transcribed (TS) or non-transcribed (NTS) strand. DNA templates were transcribed in vitro such that the transcripts were radioactively labeled. Elongation was allowed to proceed for 30 min after addition of NTPs to the reaction. Lanes 1 and 3, templates containing G-rich sequences in the transcribed strand; lanes 2 and 4, templates containing G-rich sequences in the non-transcribed strand. G4, transcripts arrested near G4-forming sequences; RO, full-length runoff transcript; M, 100-bp ladder.
FIGURE 2.
FIGURE 2.
Time course of T7 transcription of DNA templates containing G4-forming sequences in the non-transcribed strand. Templates containing G-rich sequences in either the transcribed (TS; lanes 1–7) or non-transcribed (NTS; lanes 8–14) strand were transcribed in vitro. Samples were removed from each reaction mixture at the indicated times. G4, transcripts arrested near G4-forming sequences; RO, full-length runoff transcript.
FIGURE 3.
FIGURE 3.
Effect of DNA topology on T7 transcription of DNA templates containing G-rich sequences in the transcribed or non-transcribed strand. DNA templates were transcribed as described in the legend to Fig. 1B. Lanes 1, 2, 5, and 6, templates containing G-rich sequences in the transcribed strand (TS); lanes 3, 4, 7, and 8, templates containing G-rich sequences in the non-transcribed strand (NTS). G4, transcripts arrested near G4-forming sequences; RO, full-length runoff transcript; M, 100-bp ladder.
FIGURE 4.
FIGURE 4.
RNase A and H digestion of transcripts obtained from transcription of G-rich sequences located in the transcribed or non-transcribed strand of template DNA. Lanes 1–4, templates containing G-rich sequences in the transcribed strand (TS); lanes 5–8, templates containing G-rich sequences in the non-transcribed strand (NTS). Templates were transcribed in vitro as described in the legend to Fig. 1B. Elongation was allowed for 30 min, followed by addition of RNase A (lanes 4 and 8) or RNase H (lanes 2, 3, 6, and 7) and further incubation for 30 min. RNA was then isolated and electrophoresed through a 5% polyacrylamide gel. Transcripts arrested near G4-forming sequences are indicated by G4. RO, full-length runoff transcript.
FIGURE 5.
FIGURE 5.
Effect of DNA topology on RNAP II transcription of DNA templates containing G-rich sequences in the transcribed or non-transcribed strand. Lanes 1–6, templates containing G-rich sequences in the transcribed strand (TS); lanes 7–12, templates containing G-rich sequences in the non-transcribed strand (NTS). Templates were transcribed in vitro such that transcripts were labeled with 32P as described under “Experimental Procedures.” Elongation was allowed for 30 min. RNA was then isolated and electrophoresed through a 5% polyacrylamide gel. Transcripts arrested near G4-forming sequences are indicated by G4. RO, full-length runoff transcript; M, 100-bp ladder.
FIGURE 6.
FIGURE 6.
RNase A digestion of transcripts produced after RNAP II transcription of templates containing G-rich sequences in the transcribed or non-transcribed strand. Lanes 1–5, templates containing G-rich sequences in the transcribed strand (TS); lanes 6–10, templates containing G-rich sequences in the non-transcribed strand (NTS). Templates were transcribed in vitro such that transcripts were labeled with 32P as described under “Experimental Procedures.” Elongation was allowed for 30 min, followed by addition of RNase A and further incubation for 30 min. RNA was then isolated and electrophoresed through a 5% polyacrylamide gel. Transcripts arrested near G4-forming sequences are indicated by G4. RO, full-length runoff transcript; M, 100-bp ladder (left) or 10-bp ladder (right). Triangles represent decreasing concentrations (from left to right) of RNase A.
FIGURE 7.
FIGURE 7.
Co-transcriptional formation of RNA/DNA hybrids. Lanes 1–5, templates containing G-rich sequences in the transcribed (TS) strand; lanes 6–10, templates containing G-rich sequences in the non-transcribed (NTS) strand. Templates were transcribed in vitro as described in the legend to Fig. 5. RNase A was either included in the transcription reaction (lanes 4 and 9) or added after a 30-min elongation (lanes 5 and 10). RNA was then isolated and electrophoresed through a 5% polyacrylamide gel. Transcripts arrested near G4-forming sequences are indicated by G4. RO, full-length runoff transcript; M, 100-bp ladder.
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