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. 1998;7(2):75-86.

ATP-mediated activation of RNA polymerase II transcription complexes

S J Kopytek  1 , D O Peterson
Affiliations

ATP-mediated activation of RNA polymerase II transcription complexes

S J Kopytek et al. Gene Expr. 1998.

Abstract

Transcription initiation by RNA polymerase II is a complex, multistep process that minimally involves transcription complex assembly, open complex formation, and promoter clearance. Hydrolysis of the beta--gamma phosphoanhydride bond of ATP has previously been shown to be required for open complex formation, as well as for the phosphorylation of the carboxy-terminal domain of the largest subunit of RNA polymerase II. The observation that ATP-dependent activation of transcription complexes can be blocked by ATP analogues that contain nonhydrolyzable beta--gamma phosphoanhydride bonds (such as ATPgammaS) was exploited to develop a functional kinetic assay for ATP-activated transcription complexes. Activated complexes on the promoter present in the long terminal repeat of the proviral DNA of mouse mammary tumor virus were defined as those that could productively initiate transcription in the presence of excess ATPgammaS. Activation is dependent on treatment of assembled preinitiation complexes with ATP (or dATP) prior to addition of ATPgammaS. At least 15-35% of the total number of preinitiation complexes present become activated within 2 min in the presence of (d)ATP, and activation appears to be rapidly reversible. The time course of formation of activated complexes in the presence of dATP is characterized by two kinetic phases: a rapid formation followed by a relatively slow decay. Activated complexes were estimated to form with a half-time of less than 1 min.

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Figures

FIG. 1
FIG. 1
Detection of activated pol II transcription complexes in vitro. (A) ATP-dependent activation of pol II preinitiation complexes (see text for details). (B) Requirement for dATP pretreatment to generate activated complexes. Transcription complexes were assembled on the pMBPT3 template in HeLa nuclear extract (HNE) followed by the addition of dATP (20 μM) and Sarkosyl (0.02%) at time 0. The dATP allows activation of preassembled complexes, whereas Sarkosyl prevents the assembly of any additional complexes and limits transcription to a single round. After a 2-min dATP pretreatment, [α-32P]CTP, GTP, and ATPγS (200 μM) were added, and the incubations allowed to continue for an additional 10 min (lane 1). Control reactions received no dATP (lane 3) or received dATP at the same time as the other nucleotides (lane 2). (C) Quantitation of transcription activity from complexes that received no dATP (stippled bar), dATP at the same time as the other nucleotides (open bar), or a 2-min dATP pretreatment (closed bar). Quantitation was made relative to a recovery control (RC) RNA added with the stop solution.
FIG. 2
FIG. 2
Effect of ATPγS on transcription. (A) Transcription complexes were assembled on pMBPT3 template DNA in HeLa nuclear extract (HNE). At time 0 NTPs (mix A) and Sarkosyl (0.02%) were added. In some experiments (lanes 5, 6, 9, and 10), a second NTP solution (mix B) was added after 2 min. RNA synthesis was terminated 10 min after the final addition of NTPs. All experiments contained 150 μM GTP and 50 μM (10 μCi) [α-32P]CTP added as indicated below and in the figure. Mix A contained the following nucleotides (final concentrations in the assay are indicated): CTP and GTP (lane 1); ATP (150 μM), CTP, and GTP (lane 2); dATP (10 μM), CTP, and GTP (lane 3); dATP (10 μM), ATPγS (100 μM), CTP, and GTP (lane 4); dATP (10 μM) (lanes 5 and 6); dATP (100 μM), CTP, and GTP (lane 7); dATP (100 μM), ATPγS (1 mM), CTP, and GTP (lane 8); dATP (100 μM) (lanes 9 and 10). Mix B contained the following nucleotides (final concentrations in the assay are indicated): ATPγS (100 μM), CTP, and GTP (lane 5); ATP (150 μM), CTP, and GTP (lane 6); ATPγS (1 mM), CTP, and GTP (lane 9); ATP (150 μM), CTP, and GTP (lane 10). (B) Quantitation of transcription shown in (A). Quantitation was made relative to a recovery control (RC) RNA added with the stop solution.
FIG. 3
FIG. 3
Stability of activated complexes. (A) Transcription complexes were assembled on pMBPT3 template in HeLa nuclear extract (HNE). Sarkosyl (0.02%) and dATP (20 μM) were added, and activated complexes were allowed to form for 45 s. Further activation was blocked by the addition of 10-fold molar excess ATPγS (200 μM) at time 0. Activated complexes (lanes 1–5) were observed by adding [α-32P]CTP and GTP at time t, and incubating an additional 10 min. To observe total complexes (lanes 6–9), the activation block was reversed at time t by the addition of dATP (200 μM), along with [α-32P]CTP and GTP and incubating an additional 10 min. (B) Quantitation of transcriptional activity from activated complexes (open circles) or total complexes (closed circles). Quantitation was made relative to a recovery control (RC) RNA added with the stop solution.
FIG. 4
FIG. 4
Time course of activated complex formation. (A) Preinitiation complexes were assembled on pMBPT3 template in HeLa nuclear extract (HNE) and then activated at time 0 by the addition of dATP (100 μM). Sarkosyl (0.02%) was added along with the dATP. At time I, activated complexes were allowed to transcribe by adding [α-32P]CTP, GTP, and 10-fold molar excess ATPγS (1 mM) and incubating for 10 min. (B) Quantitation of transcriptional activity from activated complexes (closed circles) and correction for (d)ATP-dependent decay (open circles) (see text for details of the correction). Quantitation was made relative to a recovery control (RC) RNA added with the stop solution. Points represent the average of at least three independent experiments, and error bars represent SEM.
FIG. 5
FIG. 5
Stability of total transcription complexes. (A) Preinitiation complexes were assembled on pMBPT3 template in HeLa nuclear extract (HNE) and then activated at time 0 by the addition of dATP (20 μM). Sarkosyl (0.02%) was added along with the dATP. At time t, ATP, [α-32P]CTP, and GTP were added and the reactions incubated an additional 10 min, thus allowing all functional transcription complexes (both activated and unactivated) to generate RNA products. (B) Quantitation of transcriptional activity from total complexes treated with dATP as described in (A) (closed circles) or from complexes that were not treated with dATP (closed squares). Quantitation was made relative to a recovery control (RC) RNA added with the stop solution. Points represent the average of at least three independent experiments, and error bars represent SEM.
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