Translocation after synthesis of a four-nucleotide RNA commits RNA polymerase II to promoter escape

Abstract

Transcription is a complex process, the regulation of which is crucial for cellular and organismic growth and development. Deciphering the molecular mechanisms that define transcription is essential to understanding the regulation of RNA synthesis. Here we describe the molecular mechanism of escape commitment, a critical step in early RNA polymerase II transcription. During escape commitment ternary transcribing complexes become stable and committed to proceeding forward through promoter escape and the remainder of the transcription reaction. We found that the point in the transcription reaction at which escape commitment occurs depends on the length of the transcript RNA (4 nucleotides [nt]) as opposed to the position of the active site of the polymerase with respect to promoter DNA elements. We found that single-stranded nucleic acids can inhibit escape commitment, and we identified oligonucleotides that are potent inhibitors of this specific step. These inhibitors bind RNA polymerase II with low nanomolar affinity and sequence specificity, and they block both promoter-dependent and promoter-independent transcription, the latter occurring in the absence of general transcription factors. We demonstrate that escape commitment involves translocation of the RNA polymerase II active site between synthesis of the third and fourth phosphodiester bonds. We propose that a conformational change in ternary transcription complexes occurs during translocation after synthesis of a 4-nt RNA to render complexes escape committed.

FIG. 1.

Escape commitment is a step in early transcription that occurs after initiation and prior to promoter escape. (A) Model depicting the steps in the RNA polymerase II transcription reaction. See the text for a description. Abbreviations: R, general transcription factors (TBP, TFIIB, TFIIF, and RNA polymerase II); P, promoter DNA (AdMLP); PIC, preinitiation complex; RP_I · (3nt RNA), initiated complex containing a 3-nt RNA; RP_EC · (4nt RNA), escape-committed complex containing a 4-nt RNA; R_E · (15nt RNA), elongation complex containing a 15-nt RNA; R_E · (FL RNA), elongation complex containing full-length RNA. (B) ctDNA inhibits escape commitment when added to reaction mixtures with the nucleotides. The method used to monitor escape commitment is depicted. ctDNA can be added at three different points in the transcription reaction: either with promoter DNA (point 1), with nucleotides (point 2), or 30 s after nucleotides (point 3). ctDNA (275 μg/ml) was added to reaction mixtures at points 2 and 3. The 390-nt G-less product is shown. (C) Escape-committed complexes contain 4-nt RNAs stably bound. The sequence of the nontemplate strand of the +5mt AdMLP is shown in boldface, with the sequence of the 4-nt RNA produced from it shown below. The schematic shows the method used to isolate ternary complexes. Transcription reactions were performed under conditions where a 4-nt RNA product is the longest that can be made at the AdMLP. Reaction mixtures were passed over G25 spin columns and phosphatase treated, and RNA products were resolved by 20% denaturing PAGE. Lane 3 is a longer exposure of lane 2. Positions of 3- and 4-nt RNA products are indicated.

FIG. 2.

Escape commitment depends on mRNA length and not on the position of the polymerase along the DNA template. (A) The schematic shows the method used to monitor escape commitment and is described in the text. (B) Escape commitment is complete after synthesis of a 4-nt RNA, regardless of where transcription initiates on the template DNA. The sequences of the nontemplate strand of the AdMLP and three mutant versions of the AdMLP are shown in boldface. The +4mt, +5mt, and +6mt promoters alter the position of the first adenosine on the nontemplate strand. At each promoter, transcription pauses at the first adenosine on the nontemplate strand in the absence of ATP. Shown on the left for each reaction are the limited nucleotides added, and the longest products formed before addition of the remaining nucleotides. The remaining nucleotides consisted of ATP, with CTP or UTP as needed to produce a full-length transcript. For each reaction ctDNA (275 μg/ml) was added either at point 2 or at point 3. The 390-nt G-less RNA product was quantitated, and the ratio of product produced when ctDNA was added at point 3 to that produced when ctDNA was added at point 2 was plotted on the graph on the right. If escape commitment occurred, the increase in transcription was ninefold or greater. Each bar represents the average of at least three measurements, and each error bar represents 1 standard deviation.

FIG. 3.

Single-stranded nucleic acid ends are required to inhibit escape commitment. (A) Method used to monitor escape commitment. Nucleic acid inhibitors were added to transcription reaction mixtures at points 1, 2, and 3. (B) Poly(dI-dC) does not inhibit escape commitment. Poly(dI-dC) and ctDNA were added to transcription reactions at the points indicated (see panel A for the method). The 390-nt G-less transcript is shown. (C) The single-stranded ends of ctDNA inhibit escape commitment. ctDNA was treated with Klenow fragment in the presence of dNTPs to remove 5′ and 3′ single-stranded overhangs and was subsequently added to assays at the points indicated. The 390-nt G-less transcript is shown.

FIG. 4.

Poly(dG) and poly(G) are potent inhibitors of escape commitment. (A) Single-stranded oligonucleotides consisting of deoxyguanosines and deoxycytidines inhibit escape commitment. The 29-dA, 29-dT, 29-dC, and 29-dG oligonucleotides (34 μM) were added to reaction mixtures at points 1, 2, and 3 (see Fig. 3A). The 390-nt G-less product is shown. (B) The IC₅₀s for the 29-dG and 29-dC oligonucleotides are 45 nM and 6.7 μM, respectively. Increasing amounts of the 29-dG and 29-dC oligonucleotides were added to transcription reaction mixtures at point 2. The 390-nt RNA was quantitated and plotted. (C) Poly(G) inhibits escape commitment. The 20rG oligonucleotide (250 nM) was added to transcription reaction mixtures at points 1 and 2. The 390-nt RNA is shown. (D) The IC₅₀ for the 20rG oligonucleotide is 12 nM. Increasing amounts of the 20rG oligonucleotide were added to transcription reaction mixtures at point 2. The 390-nt RNA was quantitated and plotted.

FIG. 5.

Single-stranded 20rG and 29-dG oligonucleotides bind RNA polymerase II and inhibit transcription in the absence of other general transcription factors. (A) The 20rG and 29-dG oligonucleotides bind directly to RNA polymerase II. A ³²P-labeled 20rG oligonucleotide (0.5 pmol) and, where indicated, unlabeled oligonucleotides (1, 5, and 50 pmol) were incubated with RNA polymerase II (35 ng). The reaction in lane 1 lacked RNA polymerase. (B) The 20rG and 29-dG oligonucleotides inhibit transcription from a poly(dA-dT) · poly(dA-dT) template DNA. RNA polymerase II was added to reaction mixtures containing poly(dA-dT), ATP, UTP, and inhibitor oligonucleotides (250 nM). Transcription proceeded for 20 min. The nonspecific transcript is shown. (C) The 20rG oligonucleotide inhibits promoter-independent transcription with an IC₅₀ of 2.3 nM. Reactions were performed as described above. The nonspecific product was quantitated and plotted as shown.

FIG. 6.

The site on RNA polymerase II to which the inhibitor oligonucleotides bind is removed from the site at which inhibition occurs. (A) An RNA oligonucleotide consisting of 10 guanosine residues (the 10rG oligonucleotide) does not inhibit escape commitment to the same extent as the 20rG oligonucleotide. The 10rG oligonucleotide was titrated into transcription reaction mixtures at point 2 (see Fig. 3A). The 390-nt RNA was quantitated and plotted. The IC₅₀ is 0.5 μM. (B) The 10rG oligonucleotide and the 20rG oligonucleotide bind to RNA polymerase II with similar affinities. Electrophoretic mobility shift assays were performed with ³²P-labeled 10rG and 20rG oligonucleotides (0.05 pmol) and increasing amounts of RNA polymerase II (3.5, 7, 14, 28, and 56 ng). (C) The 10rG oligonucleotide and the 20rG oligonucleotide form kinetically stable complexes with RNA polymerase II. RNA polymerase II (14 ng) was prebound to the ³²P-labeled oligonucleotide indicated (0.05 pmol), and a 500-fold excess (25 pmol) of unlabeled oligonucleotide was subsequently added for the times indicated above lanes 4 to 6 and 10 to 12. In lanes 3 and 9, the unlabeled oligonucleotide was added prior to RNA polymerase II.

FIG. 7.

The 29-dG oligonucleotide does not inhibit abortive initiation. (A) Schematic of the abortive-initiation assay, a steady-state assay monitoring production of 3-nt RNAs. (B) Abortive initiation was performed on the AdMLP in the absence and presence of the 29-dG oligonucleotide (250 nM). When present, the 29-dG oligonucleotide was added at point 2 (see Fig. 3A). ApCpU product was quantitated at different time points after addition of nucleotides. Each error bar represents 1 standard deviation.

FIG. 8.

Escape commitment involves translocation of the RNA polymerase II active site between synthesis of the third and fourth phosphodiester bonds. (A) Method used to isolate ternary complexes. Transcription reactions were performed in the absence and presence of the 20rG oligonucleotide (250 nM) under conditions where a 15-nt RNA product is the longest that can be made at the AdMLP. Reaction mixtures were passed over G25 spin columns and then phosphatase treated, and RNA products were resolved by 20% denaturing PAGE. (B) The 4-nt RNA product is part of a stable inhibited complex at the AdMLP. Reactions were performed as described in the text. Note that the reactions for lanes 1 and 2 were initiated with ApC while the reaction for lane 3 was initiated with ATP. Size markers and positions of 4- and 15-nt RNA products are indicated. (C) Escape-committed complexes with the 20rG oligonucleotide bound can undergo pyrophosphorolysis. Ternary complexes containing the 20rG oligonucleotide (250 nM) were isolated as described for panel A and incubated in the presence or absence of pyrophosphate (1 mM) for 30 min at 30°C. The 4-nt RNA product is shown.

FIG. 9.

Model depicting escape commitment within the context of the transcription reaction. The oligonucleotide inhibitors block translocation of the polymerase active site immediately after a 4-nt RNA is produced. All other events in transcription are resistant to the inhibitors. A detailed explanation of the model is provided in the text.