An 8 nt RNA triggers a rate-limiting shift of RNA polymerase II complexes into elongation

Abstract

To better understand the critical conversions that RNA polymerase II complexes undergo during promoter escape, we determined in vitro the precise positions of the rate-limiting step and the last step requiring negative superhelicity or TFIIE and TFIIH. We found that both steps occur after synthesis of an 8 nt RNA during the stage encompassing translocation of the polymerase active site to the ninth register. When added to reactions just before this step, TFIIE and TFIIH overcame the requirement for negative superhelicity. The positions at which both steps occur were strictly dependent on RNA length as opposed to the location of the polymerase relative to promoter elements, showing that the transcript itself controls transformations during promoter escape. We propose a model in which completion of promoter escape involves a rate-limiting conversion of early transcribing complexes into elongation complexes. This transformation is triggered by synthesis of an 8 nt RNA, occurs independent of the general transcription factors, and requires under-winding in the DNA template via negative superhelicity or the action of TFIIE and TFIIH.

Figure 1

A working kinetic model for the Pol II transcription reaction obtained using a minimal in vitro transcription system. The rate-limiting step occurs during promoter escape. R, Pol II and the general transcription factors; P, promoter DNA; PIC, preinitiation complex; RP_I, initiated complex; RP_EC, escape committed complex; R_E, elongation complex.

Figure 2

The rate-limiting step occurs after synthesis of a 7 nt RNA and before loading the ninth NTP. (A) The approach used to determine the position of the rate-limiting step during promoter escape. See text for description. (B) The rate-limiting step occurs at a specific position during early transcription. Rate constants for extension of short paused RNA transcripts to full-length transcripts on the +7, +8, +9, +10, +11, +12, and +14T promoters are plotted logarithmically and compared to those measured on the +16 and +5T promoters (dashed lines). Where error bars are shown, they represent one standard deviation and were obtained from the average of at least three experiments. The rate constants are as follows: +7T, 2.8±0.05 × 10⁻³ s⁻¹; +8T, 2.5±0.3 × 10⁻³ s⁻¹; +9T, 13±1 × 10⁻³ s⁻¹; +10T, 13±3 × 10⁻³ s⁻¹; +11T, 9.0±0.9 × 10⁻³ s⁻¹; +12T, 11 × 10⁻³ s⁻¹; +14T, 20 × 10⁻³ s⁻¹. (C) The position of the rate-limiting step is independent of the general transcription factors. Ternary complexes were assembled from Pol II, AdMLP template DNA, and RNA oligonucleotides (7 or 8 nt) as shown in the schematic, and 29 nt extended RNA transcripts were monitored. The observed ratios of 29 nt transcripts at 2 and 20 min were determined and compared to the expected ratios for extension of 7 and 8 nt RNAs calculated from the rate constants in panel B. The efficiency with which the 7 nt RNA was extended was significantly less than that for the 8 nt RNA, as was expected from published work (Kireeva et al, 2000). Therefore, the image showing extension of the 7 nt RNA was obtained from a longer exposure than that for the 8 nt RNA.

Figure 3

The last step requiring negative superhelicity occurs after synthesis of a 7 nt RNA and before loading the ninth NTP. The ratio of full-length transcript produced with and without cutting is plotted versus promoter. Each bar represents the average of at least three experiments and error bars are one standard deviation.

Figure 4

The positions of the rate-limiting step and the last step requiring negative superhelicity are set by the length of the RNA transcript. (A) Transcription can be initiated at −1 or +2 using the dinucleotide CpA or CpU, respectively. Shown are the sequences of the template strands of the +8 and +9T promoters and the paused RNA transcripts when transcription is initiated with ApC, CpA, or CpU. (B) The rate of extension of paused transcripts to full-length RNA is dictated by the length of the transcript in paused ternary complexes. The closed bars depict the rate constants for extension of paused transcripts initiated with CpA on the +8T promoter (11±0.5 × 10⁻³ s⁻¹) and CpU on the +9T promoter (2.7±0.3 × 10⁻³ s⁻¹). Rate constants are the average of two experiments and the error bars are the range of two rate constants. The open bars depict the rate constants for extension of paused transcripts initiated at +1 with ApC on the +8 and +9T promoters (from Figure 2B). (C) The position of the last step requiring negative superhelicity is dictated by RNA length. Transcription was initiated at −1 or +2 using the dinucleotide CpA or CpU, respectively. The closed bars depict the ratio of full-length transcript produced from paused ternary complexes initiated with CpA on the +8T promoter or CpU on the +9T promoter. The open bars depict the ratio of full-length transcript produced from paused ternary complexes initiated with ApC on either the +8T promoter or the +9T promoter (from Figure 3). All bars are an average of at least three experiments and error bars are one standard deviation.

Figure 5

The rate-limiting step occurs after synthesis of an 8 nt RNA. (A) The model shows the three stages of the reaction that could be rate-limiting: N, loading of the eighth NTP; C, synthesis of the seventh phosphodiester bond; and T, translocation to the ninth register. Ternary complexes denoted with an asterisk are post-translocated, whereas those lacking an asterisk are pre-translocated. (B) An 8 nt RNA is synthesized rapidly, with a rate constant of 19 × 10⁻³ s⁻¹. The +8T9C promoter and the dinucleotide CpA were used to limit read-through and slipped products. 3′-Me-GTP was not included. (C) An 8 bp RNA:DNA hybrid is not needed to complete the rate-limiting step. The nucleotides used are shown to highlight the point that the 5′ ends of RNAs initiated with ApApC were not complementary to the DNA. The rate constants for extension of 5, 7, 8, and 9 nt RNAs initiated with ApApC are plotted logarithmically. The rate constants are as follows: +5T, 2.1 × 10⁻³ s⁻¹; +7T, 2.7 × 10⁻³ s⁻¹; +8T, 11 × 10⁻³ s⁻¹; +9T, 11 × 10⁻³ s⁻¹.

Figure 6

The last step requiring negative superhelicity is coincident with the rate-limiting step. The rate at which Pol II becomes resistant to linearization is slow, with a rate constant of 3.8 × 10⁻³ s⁻¹. The diagram depicts the method used to determine the rate at which transcription becomes resistant to linearization.

Figure 7

The effect of TFIIE and TFIIH on the rate-limiting step and the requirement for negative superhelicity. (A) An 8 nt RNA is synthesized rapidly from preinitiation complexes assembled with TFIIE and TFIIH. Reactions were performed using the +9T promoter and the dinucleotide ApC. dATP (100 μM) was included in the nucleotide mix as a source of hydrolyzable nucleotide for the TFIIH helicase. The rate constant is 42 × 10⁻³ s⁻¹. (B) In the presence of TFIIE and TFIIH, 8 nt RNA is extended to full length rapidly. The rates of extension were measured in the presence of TFIIE and TFIIH for 4 and 8 nt RNAs formed on the +5 and +9T promoters, respectively. Rate constants are as follows: +5T, 5.8 × 10⁻³ s⁻¹; +9T, 28 × 10⁻³ s⁻¹. (C) TFIIE and TFIIH rescue paused ternary complexes containing 7 nt RNA on linearized DNA templates. Paused complexes containing 7 nt transcripts were formed on the +8T promoter, DNA was linearized with HindIII, TFIIE and TFIIH were added to half of the reactions, and 1 min later extension nucleotides were added. The amount of full-length RNA was quantitated. Bars are an average of at least three experiments and error bars are one standard deviation. The dashed line is the amount of full-length transcript produced in control reactions in which TFIIE and TFIIH were added to preinitiation complexes on linearized DNA templates 1 min before the pulse nucleotides.

Figure 8

A model depicting two critical transformations that occur as preinitiation complexes transform into elongation complexes. Both escape commitment and promoter escape are complete during translocation stages (T), are triggered by RNAs of distinct sizes (4 and 8 nt), and occur in the absence of general transcription factors (GTFs). Ternary complexes denoted with an asterisk are post-translocated, whereas those lacking an asterisk are pre-translocated.