RNA polymerase II complexes in the very early phase of transcription are not susceptible to TFIIS-induced exonucleolytic cleavage

Abstract

TFIIS is a transcription elongation factor for RNA polymerase II (pol II), which can suppress ribonucleotide misincorporation. We reconstituted transcription complexes in a highly purified pol II system on adenovirus Major-Late promoter constructs. We noted that these complexes have a high propensity for read-through upon GTP omission. Read-through occurred during the early stages at all registers analyzed. Addition of TFIIS reversed read-through of productive elongation complexes, which indicated that it was due to misincorporation. However, before register 13 transcription complexes were insensitive to TFIIS. These findings are discussed with respect to the structural models for pol II and we propose that TFIIS action is linked to the RNA:DNA hybrid.

Figure 1

DNA sequence of the non-template strand of the pDN-AdML+nG plasmids. Indicated in bold are the natural transcription start site (also depicted by the broken arrow) and the first three G residues in the RNA chain. Transcribed sequences are depicted in capitals and template sequences outside the indicated region are identical.

Figure 2

Analysis of the RNA products formed with the mutant AdML promoter templates in absence and presence of ΔTFIIS or full-length TFIIS. Transcription initiation complexes were assembled for 30 min on AdML+3G (lanes 1–3), AdML+4G (lanes 4–6), AdML+6G (lanes 7–9), AdML+7G (lanes 10–12), AdML+9G (lanes 13–15), AdML+10G (lanes 16–18), AdML+11G (lanes 19–21), AdML+13G (lanes 22–24), AdML+15G (lanes 25–27), AdML+17G (lanes 28–30), AdML+19G (lanes 31–33), AdML+21G (lanes 34–36), AdML+23G (lanes 37–39) or AdML+25G (lanes 40–42) templates as described in the Materials and Methods. Reactions 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37 and 40 received no TFIIS while reactions 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38 and 41 contained 40 nM ΔTFIIS protein and reactions 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39 and 42 contained 40 nM full-length TFIIS protein. Nucleotide triphosphates (60 µM ATP, 10 µM UTP and 2 µM [α-³²P]CTP) were added for 15 min. Subsequently, RNA products were processed and analyzed by denaturing polyacrylamide gel electrophoresis as described in Materials and Methods. The length of the RNA products is indicated on the left (for lanes 1–6) and right of the figure.

Figure 3

Titration of nucleotides and TFIIS protein in transcription reactions using the AdML+11G template. (A) Titration of ATP and TFIIS. Transcription initiation complexes were assembled in absence or presence of varying concentrations of full-length TFIIS protein as indicated at the top of the figure. Reactions 1–4 received no TFIIS. Reactions 5–8 received 40 nM TFIIS. Reactions 9–11 received 120, 400 and 1200 nM TFIIS, respectively. Nucleotide triphosphates (10 µM UTP, 2 µM [α-³²P]CTP and varying concentrations of ATP as indicated at the top of the figure) were added for 15 min. (B) Titration of UTP and TFIIS. Transcription initiation complexes were assembled in absence or presence of varying concentrations of full-length TFIIS proteins as indicated at the top of the figure. Reactions 1–4 received no TFIIS. Reactions 5–8 received 40 nM TFIIS. Reactions 9–11 received 120, 400 and 1200 nM of TFIIS, respectively. Nucleotide triphosphates (60 µM ATP, 2 µM [α-³²P]CTP and varying concentrations of UTP as indicated at the top of the figure) were added for 15 min. All reactions were processed and analyzed by denaturing polyacrylamide gel electrophoresis as described in Materials and Methods. The length of the RNA products is indicated on the left of the figure parts and the sequence of the non-template strand of pDNAdML+11G is indicated on the right of the figure parts with the G residues shown in bold.

Figure 4

TFIIS does not alter the kinetics of synthesis of small RNA products. Transcription initiation complexes were assembled in absence or presence of full-length TFIIS on the mutant AdML templates indicated at the top of the figure. Reactions 1–4, 9–12 and 17–20 received no TFIIS, while reactions 5–8, 13–16 and 21–24 received 40 nM full-length TFIIS protein. Initiation complexes were assembled for 30 min with AdML+3G (lanes 1–8), AdML+9G (lanes 9–16) and AdML+15G (lanes 17–24). Nucleotide triphosphates (60 µM ATP, 10 µM UTP, 2 µM [α-³²P]CTP) were added for the times indicated at the top of the figure (0.5, 1.5, 5 or 15 min). Subsequently, RNA products were processed and analyzed by denaturing polyacrylamide gel electrophoresis as described in Materials and Methods. The length of the RNA products is indicated on the left (for lanes 1–8) and right of the figure.

Figure 5

TFIIS also functions after read-through products have been formed. Transcription initiation complexes were assembled in absence or presence of 40 nM full-length TFIIS on mutant AdML templates as indicated at the top of the figure. Reactions 1 and 5 received no TFIIS, reactions 2 and 6 received TFIIS during the pre-incubation, reactions 3 and 7 received TFIIS together with the nucleotide triphosphates and reactions 4 and 8 received TFIIS 5 min after the addition of nucleotide triphosphates. Initiation complexes were assembled with AdML+6G (lanes 1–4) and AdML+15G (lanes 5–8). The nucleotide triphosphates (60 µM ATP, 10 µM UTP, 2 µM [α-³²P]CTP) were added for 15 min. Subsequently, RNA products were processed and analyzed by denaturing polyacrylamide gel electrophoresis as described in Materials and Methods. The lengths of the RNA products are indicated on the left of the figure.