Transcriptional termination and coupled polyadenylation in vitro - PubMed (original) (raw)

Transcriptional termination and coupled polyadenylation in vitro

M Yonaha et al. EMBO J. 2000.

Abstract

Using a coupled, in vitro transcription and polyadenylation system we have investigated the molecular mechanism of transcriptional termination by RNA polymerase II (PolII). We showed previously that specific G-rich sequences pause transcription and then activate polyadenylation. We show that physiological pause sites activate polyadenylation in our in vitro system. We also investigate the mechanism of PolII transcriptional termination, and show that these transcripts are either directly released from the transcription complex or are 3' end processed while still attached to the complex. We also show that 3' product (generated by cleavage/polyadenylation) remains associated with the transcription complex, but is rapidly degraded on it.

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Figures

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Fig. 1. Physiological sequences activate 3′ end processing. (A) The C2 pause site activates 3′ end processing. The wild-type C2 template and the DNA probe are indicated in the diagram. The MAZ site in the C2 pause site and the MAZ4 sequences, GGGGGAGGGGG, was mutated to GG

T

G

A

A

A

GG

T

G. Lane 1, marker; lane 2, wild-type C2 template; lane 3, mutant C2 template; lane 4, wild-type SPA and wild-type MAZ4 template; lane 5, wild-type SPA and mutant MAZ4 template. (B) A ‘roadblock’ to PolII elongation in the C2 pause site by Gln111 activates 3′ end processing. The _Eco_RI site is just downstream of the C2 pause site and the MAZ4 sites on the C2 and MAZ4 templates, respectively. The _Eco_RI site is 92 bp downstream of the poly(A) site on the T-92 template (see Figure 2, diagram). Lane 1, marker; lanes 2 and 3, wild-type C2 template; lanes 4 and 5, wild-type SPA and wild-type MAZ4 template; lanes 6 and 7, T-92 template. The reaction mixtures were preincubated with 0.05 mg of Gln111 _Eco_RI (lanes 3, 5 and 7) or not (lanes 2, 4 and 6). (C) The α pause site activates 3′ end processing. The α template and DNA probe are indicated in the diagram. Lane 1, marker; lane 2, α template; lane 3, T-92 template. (D) A ‘roadblock’ to PolII elongation in the α pause site by Gln111 activates 3′ end processing. The reaction mixture with the α template was preincubated with 0.05 mg of Gln111 _Eco_RI (lane 3) or not (lane 2). The coupled reactions were carried out for 2 h and S1 protected DNA products were analyzed on 6% polyacrylamide–8 M urea gel. < indicates a partial digestion product; << indicates a specific hybridization product from the 3′ flanking region near the end of the DNA templates (see Figure 2, diagram).

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Fig. 2. Transcripts paused at MAZ sites are resistant to sarkosyl treatment. Lane 1, marker; lanes 2, 3, 6 and 7, wild-type SPA and wild-type MAZ4 site template; lane 4, 5, 8 and 9, T-92 template. The templates are indicated in the diagram with 3′ probe. The reaction mixture with the T-92 template was preincubated with 0.05 mg of Gln111 _Eco_RI. The coupled in vitro transcription–polyadenylation reaction was initiated for 5 min at 30°C without RNase inhibitor, then 0.1% sarkosyl was added to the reactions shown in lanes 2–5 and reactions were continued. The total reaction times are indicated. S1-protected DNA products were analyzed on 6% polyacrylamide–8 M urea gel. < and << are as in Figure 1 (see the diagram).

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Fig. 3. Transcript analysis using immobilized templates. (A) Transcripts paused at MAZ sites are partially released from the ternary complex. Lane 1, marker; lane 2, wild-type SPA and wild-type MAZ4 site template; lanes 3–5, immobilized wild-type SPA and wild-type MAZ4 template (indicated in the diagram with 3′ probe); lane 6, T-92 template; lanes 7–9, immobilized T-92 template. The reaction mixture with T-92 and immobilized T-92 templates was preincubated with 0.05 mg of Gln111 _Eco_RI. The coupled in vitro transcription–polyadenylation reaction was carried out for 3 h. Transcripts with immobilized templates were then fractionated into template-bound (b) and supernatant fractions (r) by magnetic selection. The unfractionated total reaction (t) is shown. These transcripts were analyzed by S1 nuclease protection using 3′ probe on 6% polyacrylamide–8 M urea gel. (< and << are as in Figure 1). (B) Activation of polyadenylation occurs on the ternary complex. Transcripts produced during 30 min incubation with the immobilized wild-type SPA and wild-type MAZ4 template were fractionated. The bound fraction was mixed with supernatant obtained after 30 min incubation with magnetic beads. α-amanitin (2 µg/ml) was added to both fractions to halt transcription and incubated at 30°C for a further 4 h. The released fraction was supplemented with additional nuclear extract in a separate experiment (data not shown). RNAs were analyzed by S1 nuclease protection using 3′ probe on a 6% polyacrylamide–8 M urea gel. b and r are as in (A). The diagram below outlines the experimental approach. MS denotes magnetic selection.

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Fig. 4. 3′ product remains associated with the ternary complex and is rapidly degraded. (A) Coupled reactions with immobilized wild-type (lanes 2–7) or mutant (lanes 8 and 9) SPA, 118 bp spacer and wild-type MAZ4 site template were carried out for the times indicated. The AATAAA sequence of the SPA was mutated to GCGGCG. The transcripts were fractionated and analyzed by S1 nuclease protection using the 5′ probe on a 9% polyacrylamide–8 M urea gel. b and r are as in Figure 3A. (B) Analysis of the stability of the cleaved 3′ product. The wild-type (lanes 2–5 and 8–11) or mutant (lanes 6 and 7) SPA, the 118 bp spacer and wild-type MAZ4 templates were transcribed in the coupled system without RNase inhibitor for 0.5 h. Transcription was halted by addition of 2 µg/ml α-amanitin and the reactions were continued. The total reaction times are indicated. The transcripts were analyzed by S1 nuclease protection using the 5′ probe on a 9% polyacrylamide–8 M urea gel. Lanes 8–11 are longer exposures of lanes 2–5, respectively. (C) Comparison of stability of the 5′ and 3′ products. The same reaction as in (B) with wild-type SPA, 118 bp spacer and wild-type MAZ4 template. Transcripts were analyzed by S1 nuclease protection using the 3′ (lanes 2–4) or 5′ probe (lanes 5–7) on a 7.5% polyacrylamide–8 M urea gel. The DNA templates and the DNA probes used are indicated in the diagram. << is as in Figure 1. Note that the read-through product detected with the 5′ and 3′ probes used in Figure 4 will include all RNAs that have not cleaved at SPA.

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Fig. 5. Diagram illustrating the different steps associated with PolII termination. Steps A–C were directly investigated in these studies.

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