Comparison of traveling ratios for wild-type and ΔFL RNAPII on genes with partial or strong promoter escape. (A) ΔFL ettRNAPII versus wild-type ettRNAPII. Data are color coded gray for partial promoter escape (TR, <0.49), black for strong promoter escape (TR, >0.49), and blue (TR, <0.49) or red (TR, >0.49) for genes in ENCODE regions. (B) RPB1 signals versus wild-type ettRNAPII signals. The color scheme is the same as that in panel A.

Wild-type and ΔFL ettRNAPII are transcriptionally active in vivo. (A) Map of chromosome 10 (chr 10) showing the location of the randomly selected C10orf28 gene. Log₂ ratios of IP to input signals from RPB1, wild-type ettRPB2, and ΔFL ettRPB2 samples, and the ratio of wild-type ettRPB2 to ΔFL ettRPB2 at the orf28 gene, are shown. (B to D) Scatter plots of RPB1, Flag RPB2 WT, and Flag RPB2 ΔFL signals. The signal for each probe is plotted against those of the other probes.

The flap loop does not inhibit backtracking. (A) Reaction scheme for TFIIS-stimulated transcript cleavage. The sequence of the nucleic acid scaffold used to reconstitute ECs is shown with the position of the halted A24 EC indicated. (B) A24 complexes made in the presence of ATP and GTP were incubated with apyrase to convert NTPs to NDPs. The ECs were then incubated with TFIIS; aliquots were removed at the times indicated; and the RNA products were separated on a 20% denaturing polyacrylamide gel. The fraction of remaining A24 RNA is plotted as a function of reaction time.

Distribution of ChIP signals or CMARRT regions relative to TSSs. The regions tiled in the array were classified into subsets, eliminating regions of <10 kb and >500 kb. The distance of the nearest TSS was calculated for each of the IPs and was plotted as a function of distance upstream or downstream from the nearest promoter. If a promoter was present within a CMARRT region, then the distance from the promoter to the region was assigned as zero. The total number of signals used for each condition to generate each plot is presented in Table S2 in the supplemental material. The nearest promoter was identified as described in Materials and Methods. (A) WT; (B) ΔFL; (C) RPB1; (D) WT/ΔFL ratio; (E) ΔFL/RPB1 ratio; (F) WT/RPB1 ratio.

The flap loop modestly affects transcript elongation but not TFIIF-accelerated elongation. (A) Reaction scheme and tailed-template structure, with halt and pause sites indicated. The time course of transcript elongation from halted +34 complexes was determined on the tailed template as described in Materials and Methods. Aliquots were removed at the indicated times, and RNA products were separated on a 10% denaturing polyacrylamide gel. The positions of the halted complex RNA and pause RNA (U82) are indicated next to the gel. RO, runoff. (B) The RNA in each lane was quantitated, and the fraction of the RNA longer than the U82 pause RNA was plotted as a function of reaction time. (C) Transcription elongation reactions were performed as in panel A in the absence or presence of TFIIF (10 nM). The fraction of runoff RNA was quantitated and plotted as a function of reaction time.

The flap loop is not required for transcription initiation or promoter clearance in vitro. (A) Schematic representation of preinitiation complex formation and transcription initiation from the adenovirus major late promoter (ADMLp) by wild-type and ΔFL ettRNAPII. (B) Preinitiation complexes were assembled using wild-type or ΔFL ettRNAPII with a 30-min preincubation at 30°C. Transcription was initiated by the addition of NTPs for the indicated times, and RNA products were separated on a 20% denaturing polyacrylamide gel. The fractions of 4-mer and 23-mer RNAs were quantitated and plotted as a function of reaction time. (C) Preinitiation complexes were assembled using wild-type or ΔFL ettRNAPII without preincubation at 30°C. Transcription was initiated by the addition of NTPs; an aliquot was removed after the indicated time intervals; and RNA products were separated on a 20% denaturing polyacrylamide gel. The fractions of 4-mer and 23-mer RNAs were quantitated and plotted as a function of reaction time. (D and E) Ratio of the 23-mer to the 4-mer synthesized with a 30-min preincubation (D) or with no preincubation (E).

The flap loop does not contribute to DSIF/NELF-mediated inhibition of transcript elongation. (A) Sequence of the nucleic acid scaffold used to reconstitute ECs. The positions of the halted A24 EC and the runoff transcript (RO) are indicated. (B) (Left) A24 ECs made with wild-type or ΔFL ettRNAPII were elongated in the absence or presence of DSIF/NELF (D/N) and in the presence of all 4 NTPs (10 μM each). Aliquots were removed at predetermined time intervals, and RNA was separated on a 15% denaturing polyacrylamide gel. (Center) Densitometric plot of the distribution of RNA species in the lanes representing the 15-s time point in the absence (solid lines) or presence (dashed lines) of DSIF/NELF. (Right) Bar graph showing the fractions of the 24-mer and runoff RNAs for WT or ΔFL ettRNAPII in the absence (solid bars) or presence (shaded bars) of DSIF/NELF.

Wild-type and ΔFL RNAPII behave similarly at all promoters in vivo. Promoters were classified into subsets based on the wild-type RNAPII ChIP signals in a 1-kb promoter region centered at the transcription start site (TSS) (promoter signal, designated a in panel A) and in a 1-kb intragenic region between 2 and 3 kb downstream of the promoter (signal within gene, designated b in panel A). The TR was calculated as b/a separately for each TSS on the array. The IP/input signals for each class were averaged and plotted as a function of distance from the TSS. The hatched boxes in panels E and F represent a theoretical promoter and gene extending to 3 kb. Each panel contains averaged ChIP signals for wild-type ettRNAPII (blue) (anti-Flag MAb), ΔFL ettRNAPII (green) (anti-Flag MAb), and all RNAPII (red) (anti-RPB1 CTD MAb). (A) Minimal or no promoter occupancy by RNAPII (promoter signal for wild-type ettRNAPII, <2). (B) Little or no promoter escape (signal within gene for wild-type ettRNAPII, <1.5). (C) Partial promoter escape (for wild-type ettRNAPII, the signal within the gene was ≥1.5 and the TR was <0.49). (D) Strong promoter escape (for wild-type ettRNAPII, the signal within the gene was ≥1.5 and the TR was ≥0.49 and <1). (E and F) Full ChIP signal profiles for genes in ENCODE regions that exhibited partial promoter escape (for wild-type ettRNAPII, the signal within the gene was ≥1.5 and the TR was <0.49) (E) and strong promoter escape (for wild-type ettRNAPII, the signal within the gene was ≥1.5 and the TR was ≥0.49 and <1) (F). See Materials and Methods for a description of the averaging method.

Conditional expression, partial purification, and in vitro transcriptional activities of wild-type and ΔFL ettRNAPII. (A) Conditional expression of wild-type and ΔFL ettRNAPII in HEK 293 cells. Cell lysates from cultures induced with doxycycline (2 μg/ml) were immunoprecipitated with either anti-Flag antibody M2 (lanes 1 to 4) or RPB1-specific 8WG16 antibodies (lanes 5 to 8). The immunoprecipitated proteins were fractionated on an SDS–4 to 12% polyacrylamide gel and were detected by immunoblotting. Anti-Flag antibodies were used to detect the expression of the ettRPB2 subunit and its assembly into RNAPII. (B) Partial purification of wild-type and ΔFL ettRNAPII. Flag-tagged wild-type and ΔFL RNAPII were affinity purified on anti-Flag M2 agarose, fractionated on an SDS–4 to 16% polyacrylamide gel, and stained with silver. Purified calf thymus (CT) RNAPII served as a marker. The positions of the 12 subunits of RNAPII are indicated. (C) Sequences of the DNA and RNA oligonucleotides used to reconstitute ECs. The positions of the halted EC18 and EC24 are indicated. Transcription elongation complex reconstitution, EC18 and EC24 formation, and immobilization of ECs on Ni²⁺ agarose beads are shown schematically. (D) Purified wild-type and ΔFL ettRNAPII are transcriptionally active and are not contaminated with endogenous wild-type RNAPII. Reconstituted wild-type EC16 and ΔFL ettRNAPII EC16 were elongated in the presence of ATP and GTP to make G24 ECs (ettEC24). Control ctEC18 was made using purified CT RNAPII and was mixed with either wild-type or ΔFL ettRNAPII. The complexes were then immobilized on Ni²⁺ agarose beads and were separated by centrifugation, and the RNA in the total (Tot) and supernatant (Sup) fractions was separated on a 20% denaturing polyacrylamide gel. (E) The fractions of ettEC24 and ctEC18 bound to Ni²⁺ agarose beads were determined, establishing that >90% of the transcriptional activity in the ettRNAPII preparations was derived from the tagged enzymes and not from native, untagged RNAPII, which could potentially contaminate the preparations.

Expression of wild-type and ΔFL ettRNAPII. (A) Structure of Saccharomyces cerevisiae RNAPII in complex with TFIIB (Protein Data Bank identification code [PDB ID] 3k1f) (26) with the flap loop (blue/green) enlarged in the inset. The flap loop deletion is shown in green. (B) Schematic diagram of the epitope-tagged human RPB2 gene. The sequence alignment of the human flap loop (blue) is shown with the deletion boxed in green. The flap loop in S. cerevisiae RNAPII is shown in blue, and the flap loop helix is indicated by a helix above the sequence. The Thermus thermophilus and E. coli flap tip sequences are shown in black, with the flap tip helix boxed and indicated by a helix above the sequence. The locations of the E. coli flap tip (ΔFT) and flap tip helix (ΔFTH) deletions studied previously (43, 44) are indicated. SV40, simian virus 40; H sa, Homo sapiens; S ce, S. cerevisiae; T th, T. thermophilus; E co, E. coli. (C) The epitope tag is genetically linked to the flap loop deletion in the genome. The structure of the integrated transgene, the locations of the primers, and the sizes of the amplicons are shown schematically. Genomic DNA was first amplified using primer set 1/4 (lanes 1 to 4) from the control plasmid DNA (cpDNA) or from genomic DNA from the transgenic cell line (tcDNA), and the gel-purified 3-kb fragment was used as a template for the second round of PCR using primer set 1/2 (lanes 6, 7, 12, and 13), 3/4 (lanes 8 to 11), or 5/4 (lanes 14 to 21). The WT or ΔFL plasmids used for transfection served as positive-control templates (cpDNA). Samples were separated on a 1% (lanes 1 to 4) or a 1.5% (lanes 5 to 21) agarose gel. (D) The mRNA that codes for the epitope tags is linked to the flap loop deletion. The structure of mRNA coding for the transgene, the locations of the primers, and the sizes of the amplicons are shown schematically. cDNA was synthesized from total RNA using primer 4, and a 3-kb DNA fragment was PCR amplified from the cDNA or control plasmids (cpDNA) using primer set 1/4 (lanes 1 to 4). The gel-purified 3-kb amplicon was then used as a template for a second round of PCR using primer set 1/2 (lanes 5 to 8) or 3/4 (lanes 9 to 14). The WT or ΔFL plasmids used for transfection served as positive-control templates (cpDNA). Samples were separated on a 1% (lanes 1 to 4) or a 1.5% (lanes 5 to 14) agarose gel.