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. 2002 Jan;22(1):30-40.
doi: 10.1128/MCB.22.1.30-40.2002.

Strong natural pausing by RNA polymerase II within 10 bases of transcription start may result in repeated slippage and reextension of the nascent RNA

Mahadeb Pal  1 , Donal S Luse
Affiliations

Strong natural pausing by RNA polymerase II within 10 bases of transcription start may result in repeated slippage and reextension of the nascent RNA

Mahadeb Pal et al. Mol Cell Biol. 2002 Jan.

Abstract

We find that immediately following transcript initiation, RNA polymerase II pauses at several locations even in the presence of relatively high (200 microM) levels of nucleoside triphosphates. Strong pauses with half-lives of >30 s were observed at +7, +18/19, and about +25 on the template used in these experiments. We show that the strong pause at +7, after the synthesis of 5'-ACUCUCU, leads to repeated cycles of upstream slippage of the RNA-DNA hybrid followed by re-pairing with the DNA and continued RNA synthesis. The resulting transcripts are 2, 4, and 6 bases longer than predicted by the template sequence. Slippage is efficient when transcription is primed with the +1/+2 (ApC) dinucleotide, and it occurs at even higher levels with the +2/+3 primer (CpU). Slippage can occur at high levels with ATP initiation, but priming with CpA (-1/+1) supports very little slippage. This latter result is not simply an effect of transcript length at the point of pausing. Slippage can also occur with a second template on which the polymerase can be paused after synthesizing ACUCU. Slippage is not reduced by an ATP analog that blocks promoter escape, but it is inhibited by substitution of 5Br-U for U in the RNA. Our results reveal an unexpected flexibility of RNA polymerase II ternary complexes during the very early stage of transcription, and they suggest that initiation at different locations within the same promoter gives rise to transcription complexes with different properties.

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Figures

FIG. 1.
FIG. 1.
Sequences of the nontemplate strands of the initially transcribed regions of the plasmids used in these studies. The single G residue which differs from the pML20-40 sequence in the 4G, 6G, and 9G templates is shown in each instance in lowercase type.
FIG. 2.
FIG. 2.
RNA polymerase II pauses at distinct locations in the early phase of RNA synthesis on the pML20-40 template. (A) RNA synthesis performed at 30°C with 1 mM ApC and 200 μM each ATP, GTP, CTP, and UTP. The CTP and UTP were each 32P labeled (final specific activity, 40 Ci/mmol). The reactions were stopped at the indicated times by pipetting the mixtures directly into phenol-chloroform-isoamyl alcohol. (B) RNA synthesis performed at 25°C with 1 mM ApC, 20 μM each CTP, GTP, and dATP, and 2 μM [α-32P]UTP. Transcripts longer than predicted by the template sequence are marked by asterisks. For both panels, lanes marked α-am contained 2 μg of α-amanitin per ml. Solid arrows indicate sites of pausing.
FIG. 3.
FIG. 3.
Transcript slippage on the pML20-40 template. (A) In lanes 1 to 3, RNA was synthesized with 1 mM ApC, 2 μM [α-32P]UTP, and 20 μM each CTP, GTP, and dATP at 30°C for 4 min; 20 μM nonlabeled UTP was then added, and the reaction was continued for 4 min. Transcripts were purified (lane 1) and then digested with 1 U (lane 2) or 2 U (lane 3) of RNase T1 per 10 μl of reaction mixture. Length standards were generated by pyrophosphorolysis (PPi) of transcripts halted at C27 or U20 on template pML20-40 (4G). The single 4-nt T1 cleavage product (5′-CUUU-3′) from the 3′ termini of the RNAs is indicated (lanes 2 and 3). The slippage-reextension products are marked by asterisks. (B) Schematic of the first and second round of transcript slippage and reextension on the pML20-40 template. TS, template strand; base pairing of the transcripts before and after slipping is indicated.
FIG. 4.
FIG. 4.
Under free-running transcription conditions on the pML20-40 template, transcript slippage occurs mostly from complexes paused at +7. RNA was synthesized on the indicated templates (WT is pML20-40) with ApC primers using the same protocol given in the legend of Fig. 3. One-half of each sample was digested with RNase T1. Slippage products are indicated by asterisks. Open arrowheads indicate T1 cleavage products; lengths are given to the right of the gel for the pML20-40 transcripts and to the left of the gel for the 4G, 6G, and 9G transcripts. Lane 9 (M) shows length standards generated by pyrophosphorolysis of A-stop RNA (U20 and longer slippage products) formed on the pML20-40 template. Note that transcripts of identical length from the pML20-40 and 4G, 6G, and 9G templates are expected to migrate slightly differently due to different sequence.
FIG. 5.
FIG. 5.
The initiating dinucleotide can strongly influence the efficiency of transcript slippage. Reactions used the pML20-40 template and ApC or CpA primers at 1 mM in the indicated lanes. Other transcription conditions were those given in the legend of Fig. 3, except that no GTP was added in lanes 3 to 5. Transcripts were digested with RNase T1 as indicated. Transcripts generated by slippage in lanes 1 and 5 are marked by asterisks. Lengths of nondigested ApC-primed RNAs made in the presence of GTP are given to the left of the gel, and lengths of the ApC-primed (C15, C17, and C19) and CpA-primed (C16, C18, and C20) RNAs made in the absence of GTP are given to the right of the gel. An apparently 21-nt RNA, presumed to be generated by readthrough of the G-stop in the CpA primed reaction, is indicated by (#). This was not confirmed by T1 digestion for in this particular experiment. See Fig. 6 and 7B for other examples of the low level of CpA-primed slippage and Fig. 7 for other examples of readthrough products.
FIG. 6.
FIG. 6.
Transcript slippage by RNA polymerase II paused at +5. Transcription was performed on the 6G or pML20-40 (WT) templates as indicated. Reactions contained 1 mM either ApC or CpA (as indicated) plus 2 μM [α-32P]UTP and 20 μM CTP and dATP. For the reactions in lanes 1 and 2, 6 and 7, and 11 and 12, 20 μM GTP was also added and transcription proceeded for 4 min at 30°C. Reactions in lanes 3 to 5 and 8 to 10 proceeded for 3 min without GTP: 20 μM GTP was then added, and the mixtures were incubated for a further 4 min. RNAs in the indicated lanes were digested with RNase T1. The much shorter T1 digestion products from the 6G template are not shown here (see Fig. 4). The lengths of the nondigested RNAs made on pML20-40 in the presence of GTP are given to the left of the gel. Slippage products are marked with asterisks.
FIG. 7.
FIG. 7.
Transcript initiation with the +2/+3 primer CpU promotes efficient slippage. (A) The 6G template was transcribed at 25°C with 1 mM CpU, 2 μM [α-32P]UTP, 5 μM dATP, and 20 μM CTP; reactions were stopped at the indicated times (lanes 3 to 8). Reactions in lanes 1 and 2 were done without the CpU primer. An aliquot of the 240-s reaction mixture was digested with RNase T1 (lane 8). Another aliquot of the 240-s reaction mixture was chased with 200 μM each NTP (lane 9); RNA from an equivalent reaction mixture was digested with T1 (lane 10). Slippage products are marked with asterisks, and the lengths of these RNAs are indicated to the left of the gel. (B) Transcription on the 6G template was initiated either with 1 mM dinucleotide or with 0.1 mM ATP, as indicated on the figure, along with 2 μM [α-32P]UTP, 20 μM CTP, and, in dinucleotide-containing reactions, 5 μM dATP, at 30°C for 2 min. One-half of each reaction mixture was digested with RNase T1 as indicated. For lanes 9 and 10, an ApC-primed reaction mixture otherwise identical to that in lane 3 was supplemented with 20 μM GTP; one-half of this reaction mixture was also digested with T1 as indicated (lane 10). Slippage products are marked by asterisks; transcripts resulting from readthrough of the G stop are marked by #. The lengths of the nonslipped G-stop RNAs for the CpA-, ApC-, and ATP-initiated reactions are marked on the gel. (C) The pML20-40 template was transcribed with 1 mM dinucleotide or 1 mM ATP plus 800 μM each CTP and UTP and 15 μM each [α-32P]CTP and [α-32P]UTP at 30°C for 10 min. Reaction products were ethanol precipitated, and the indicated samples were digested with RNase T1. Slippage products and RNAs resulting from readthrough of the G stop are marked by asterisks and #, respectively.
FIG. 8.
FIG. 8.
Effects of 5-Br UTP and ATPγS on transcript slippage. (A) RNA synthesis was initiated on the pML20-40 template at 25°C using 1 mM ApC, 20 μM dATP, 2 μM [α-32P]CTP, 20 μM GTP, and either 20 μM UTP or 20 μM 5Br-UTP (as marked) and continued for the indicated times. The lengths of the major paused RNAs are indicated by solid arrowheads, as well as the lengths of the A-stop RNA and slippage transcripts. Slippage products are indicated by asterisks. (B) Transcription was carried out on the 6G template with 1 mM ApC, 5 μM dATP, 2 μM [α-32P]UTP, and 20 μM CTP for 40 s at 30°C (lane 1). Aliquots of this reaction mixture were then made 20 μM in nonlabeled UTP, with (lanes 4 and 5) or without (lanes 2 and 3) 100 μM ATPγS; incubation was continued at 30°C for an additional 2 min. One-half of each of these reaction mixtures was treated with RNase T1 as indicated. To other aliquots of the 40-s reaction mixture (lane 1), 20 μM GTP was added without (lanes 6 and 7) or with (lanes 8 and 9) 100 μM ATPγS. One-half of each of these reaction mixtures was also treated with T1. Slippage products are indicated by asterisks, and RNAs produced by readthrough of the G-stop are marked by #.
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References

    1. Artsimovitch, I., and R. Landick. 1998. Interaction of a nascent RNA structure with RNA polymerase is required for hairpin-dependent transcriptional pausing but not for transcript release. Genes Dev. 12:3110–3122. - PMC - PubMed
    1. Borukhov, S., V. Sagitov, C. A. Josaitis, R. L. Gourse, and A. Goldfarb. 1993. Two modes of transcription initiation in vitro at the rmB P1 promoter of Escherichia coli. J. Biol. Chem. 268:23477–23482. - PubMed
    1. Cowie, A., P. Jat, and R. Kamen. 1982. Determination of sequences at the capped 5′ ends of polyoma virus early region transcripts synthesized in vivo and in vitro demonstrates an unusual microheterogeneity. J. Mol. Biol. 159:225–255. - PubMed
    1. Dedrick, R. L., C. M. Kane, and M. J. Chamberlin. 1987. Purified RNA polymerase II recognizes specific termination sites during transcription in vitro. J. Biol. Chem. 262:9098–9108. - PubMed
    1. Dvir, A., J. W. Conaway, and R. C. Conaway. 2001. Mechanism of transcription initiation and promoter escape by RNA polymerase II. Curr. Opin. Genet. Dev. 11:209–214. - PubMed

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