Promoter clearance by RNA polymerase II is an extended, multistep process strongly affected by sequence.

We have characterized RNA polymerase II complexes halted from +16 to +49 on two templates which differ in the initial 20 nucleotides (nt) of the transcribed region. On a template with a purine-rich initial transcript, most complexes halted between +20 and +32 become arrested and cannot resume RNA synthesis without the SII elongation factor. These arrested complexes all translocate upstream to the same location, such that about 12 to 13 bases of RNA remain in each of the complexes after SII-mediated transcript cleavage. Much less arrest is observed over this same region with a second template in which the initially transcribed region is pyrimidine rich, but those complexes which do arrest on the second template also translocate upstream to the same location observed with the first template. Complexes stalled at +16 to +18 on either template do not become arrested. Complexes stalled at several locations downstream of +35 become partially arrested, but these more promoter-distal arrested complexes translocate upstream by less than 10 nt; that is, they do not translocate to a common, far-upstream location. Kinetic studies with nonlimiting levels of nucleoside triphosphates reveal strong pausing between +20 and +30 on both templates. These results indicate that promoter clearance by RNA polymerase II is at least a two-step process: a preclearance escape phase extending up to about +18 followed by an unstable clearance phase which extends over the formation of 9 to 17 more bonds. Polymerases halted during the clearance phase translocate upstream to the preclearance location and arrest in at least one sequence context.

Initially transcribed sequences strongly affect the extent of abortive initiation by RNA polymerase II.

We investigated transcript initiation and early elongation by RNA polymerase II using templates mismatched between -9 and +3 (bubble templates). Highly purified RNA polymerase II alone was able to initiate transcription specifically on these templates in the presence of dinucleotide primers. The length distribution of abortively initiated RNAs was similar for purified RNA polymerase II on bubble templates and polymerase II on double-stranded templates in HeLa nuclear extracts. Increasing the U content in the initial portion of the transcript caused similar increases in abortive initiation for transcription of bubble templates by pure polymerase and double-stranded templates in extracts. Thus, the level of abortive initiation by RNA polymerase II is at least partly determined by interactions of the polymerase with the transcript and/or the template, independent of the general transcription factors. Substitution of 5-bromo-UTP for UTP reduced abortive initiation on bubble templates, consistent with the idea that transcription complex stability during early elongation depends on the strength of the initial RNA-DNA hybrid. Interestingly, transcription of bubble templates in HeLa extracts gave very high levels of abortive initiation, suggesting that inability to reanneal the initially melted template segment inhibits transcript elongation in the presence of the initiation factors.

Structural changes in the RNA polymerase II transcription complex during transition from initiation to elongation.

We obtained exonuclease III (exoIII) footprints for a series of RNA polymerase II transcription complexes stalled between positions +20 to +51. Downstream advance of the exoIII footprint is normally tightly coordinated with RNA synthesis. However, arrested RNA polymerases slide back along the template, as indicated by exoIII footprints in which the last transcribed base is abnormally close to the downstream edge of the footprint. None of the polymerase II complexes stalled between +20 and +51 were arrested. Nevertheless, the exoIII footprints of complexes with 20-, 23-, or 25-nucleotide RNAs resembled those of arrested complexes, with the last transcribed base very close to the footprint's front edge. The exoIII footprint of the +27 complex was displaced downstream by 17 bp compared to the footprint of the +25 complex. Many complexes between +27 and +42 also showed evidence of sliding back along the template. We compared the effects of template sequence and transcript length by constructing a new template in which the initial transcribed sequence was duplicated beginning at +98. The exoIII footprints of transcription complexes stalled between +122 to +130 on this DNA did not resemble those of arrested complexes, in contrast to the footprints of analogous complexes stalled over the same DNA sequences early in transcription. Our results indicate that the RNA polymerase II transcription complex passes through a major, sequence-independent structural transition about 25 bases downstream of the starting point of transcription. The fully mature form of the elongation complex may not appear until more than 40 bonds have been made.

FACT, a factor that facilitates transcript elongation through nucleosomes.

The requirements for transcriptional activation by RNA polymerase II were examined using chromatin templates assembled in vitro and a transcription system composed of the human general transcription factors and RNA polymerase II. Activator-induced, energy-dependent chromatin remodeling promoted efficient preinitiation complex formation and transcription initiation, but was not sufficient for productive transcription. Polymerases that initiated transcription on remodeled chromatin templates encountered a block to transcription proximal to the promoter. Entry into productive transcription required an accessory factor present in HeLa cell nuclear extract, FACT (facilitates chromatin transcription), which we have purified. FACT acts subsequent to transcription initiation to release RNA polymerase II from a nucleosome-induced block to productive transcription. The biochemical properties and polypeptide composition of FACT suggest that it is a novel protein factor that facilitates transcript elongation through nucleosomes.

The H3/H4 tetramer blocks transcript elongation by RNA polymerase II in vitro.

We have investigated transcript elongation efficiency by RNA polymerase II on chromatin templates in vitro. Circular plasmid DNAs bearing purified RNA polymerase II transcription complexes were assembled into nucleosomes using purified histones and transient exposure to high salt, followed by dilution and dialysis. This approach resulted in nucleosome assembly beginning immediately downstream of the transcription complexes. RNA polymerases on these nucleosomal templates could extend their 15- or 35-nucleotide nascent RNAs by only about 10 nucleotides in 15 min, even in the presence of elongation factors TFIIF and SII. Efficient transcript elongation did occur upon dissociation of nucleosomes with 1% sarkosyl, indicating that the RNA polymerases were not damaged by the high salt reconstitution procedure. Since the elongation complexes were released by sarkosyl but not by SII, these complexes apparently did not enter the arrested conformation when they encountered nucleosomes. Surprisingly, elongation was no more efficient on nucleosomal templates reconstituted only with H3/H4 tetramers, even in the presence of elongation factors and/or competitor DNA at high concentration. Thus, in a purified system lacking nucleosome remodeling factors, not only the core histone octamer but also the H3/H4 tetramer provide an nearly absolute block to transcript elongation by RNA polymerase II, even in the presence of elongation factors.

Translocation and transcriptional arrest during transcript elongation by RNA polymerase II.

RNA polymerase II may stop transcription, or arrest, while transcribing certain DNA sequences. The molecular basis for arrest is not well understood, but a connection has been suggested between arrest and a transient failure of the polymerase to translocate along the template. We have investigated this question by monitoring the movement of RNA polymerase II along a number of templates, using exonuclease III protection as our assay. We found that normal transcription is accompanied by essentially coordinate movement of the active site and both the leading and trailing edges of the polymerase. However, as polymerase approaches an arrest site, translocation of the body of the polymerase stops while transcription continues, leading to an arrested complex in which the 3' end of the transcript is located much closer than normal to the front edge of the polymerase. Surprisingly, mutated arrest sites that no longer block transcription continue to direct the transient failure of polymerase translocation. As transcription proceeds through these sequences, the initially stationary polymerase moves forward 10-15 bases along the template in response to the addition of only 3 bases to the nascent RNA. Mutagenesis studies indicate that the sequences responsible for the transient block to polymerase movement are located downstream of the T-rich motif required for arrest. Our results indicate that blocking translocation is not sufficient to cause arrest.

Amanitin greatly reduces the rate of transcription by RNA polymerase II ternary complexes but fails to inhibit some transcript cleavage modes.

The toxin alpha-amanitin is frequently employed to completely block RNA synthesis by RNA polymerase II. However, we find that polymerase II ternary transcription complexes stalled by the absence of NTPs resume RNA synthesis when NTPs and amanitin are added. Chain elongation with amanitin can continue for hours at approximately 1% of the normal rate. Amanitin also greatly slows pyrophosphorolysis by elongation-competent complexes. Complexes which are arrested (that is, which have paused in transcription for long periods in the presence of excess NTPs) are essentially incapable of resuming transcription in the presence of alpha-amanitin. Complexes traversing sequences that can provoke arrest are much more likely to stop transcription in the presence of the toxin. The substitution of IMP for GMP at the 3' end of the nascent RNA greatly increases the sensitivity of stalled transcription complexes to amanitin. Neither arrested nor stalled complexes display detectable SII-mediated transcript cleavage following amanitin treatment. However, arrested complexes possess a low level, intrinsic transcript cleavage activity which is completely amanitin-resistant; furthermore, pyrophosphorolytic transcript cleavage in arrested complexes is not affected by amanitin.

GAL4-VP16 stimulates two RNA polymerase II promoters primarily at the preinitiation complex assembly step.

We previously demonstrated that RNA polymerase II promoters may be limited in strength not only at the step of transcription complex assembly, but also at initiation or promoter clearance. Here we report on experiments designed to test the possibility that steps following transcription complex assembly might be stimulated by transcriptional activators. Using an in vitro system in which we can independently measure the efficiency of assembly, initiation, and promoter clearance, we have investigated the mechanism by which the model activator GAL4-VP16 increases transcription from two promoters: a weak variant of Ad 2 ML with an altered TATA box, which is inefficient in transcription initiation, and the mouse beta-globin promoter, which is inefficient in promoter clearance. We found that whereas GAL4-VP16 is effective in stimulating both promoters, this increase resulted only from greater transcription complex assembly; the initiation and clearance steps were not affected. Because recent studies have suggested that the core transcription factors TFIIE and TFIIH might be important in promoter clearance, we also attempted to increase the initiation and clearance efficiencies of the Ad ML-TATA mutant and globin promoters by direct addition of excess TFIIE and TFIIH to partially purified preinitiation complexes assembled at each of these promoters. These factors had no effect on transcription by either of the preinitiation complexes.

RNA polymerase II ternary complexes may become arrested after transcribing to within 10 bases of the end of linear templates.

In the presence of elongation factor SII, arrested RNA polymerase II ternary complexes cleave 7-17 nucleotides from the 3'-ends of their nascent RNAs. It has been shown that transcription of linear templates generates apparent run-off RNAs, which are nevertheless truncated upon incubation with SII. By using high resolution gels, we demonstrate that transcription of blunt or 3'-overhung templates with RNA polymerase II generates two populations of ternary complexes. The first class pauses 5-10 bases prior to the end of the template strand. These complexes respond to SII by cleaving approximately 9-17 nucleotide RNAs from their 3'-ends and therefore may be termed arrested. A second class of complexes, which fail to respond to SII, transcribe to within 3 bases of the end of the template strand. These complexes appear to have run off the template since they have released their nascent RNAs. Run-off transcription occurs on all types of templates, but it is the predominant reaction on DNAs with 5'-overhung ends. Thus, RNA polymerase II ternary complexes that retain 5-10 bases of contact with the template strand down-stream of the catalytic site become arrested. Further reduction of downstream template contacts can lead to termination. We also show that the addition of Sarkosyl to the elongation reactions significantly changes the pattern of transcriptional arrest near the end of linear templates.

Two members of the HNF-3 family have opposite effects on a lung transcriptional element; HNF-3 alpha stimulates and HNF-3 beta inhibits activity of region I from the Clara cell secretory protein (CCSP) promoter.

Region I of the Clara cell secretory protein (CCSP; also called CC10) promoter contains at least three functional factor binding sites, an upstream HNF-3 site and a downstream overlapping AP-1/HNF-3 site (Sawaya, P.L., Stripp, B. R., Whitsett, J.A., and Luse, D. S. Mol. Cell. Biol. (1993) 13, 3860-3871). Fragments containing -320/+58 of the rat CCSP promoter were mutagenized to eliminate one or two factor binding sites in region I, cloned into a luciferase reporter cassette, and assayed for activity by transfection into cultured lung (H441) cells. We found that the HNF-3 sites alone can account for the activity of region I in H441 cells. The activity of the two HNF-3 sites is synergistic; this effect depends on the presence of an upstream factor binding site. We had shown previously that H441 cells contain exclusively the HNF-3 alpha form of HNF-3, whereas HeLa cells have essentially no HNF-3. Co-transfection of an HNF-3 alpha expression plasmid with a CCSP reporter containing four copies of region I in HeLa cells stimulated CCSP activity 4-fold, whereas co-expression of HNF-3 beta inhibited activity 8-fold. HNF-3 beta was also inhibitory to region I expression in H441 cells, but to a lesser extent than in HeLa cells, presumably because of the high levels of HNF-3 alpha already present in H441 cells. We have thus identified a gene regulatory element through which two members of the HNF-3 transcription factor family, HNF-3 alpha and HNF-3 beta, exert opposite effects.

The active site of RNA polymerase II participates in transcript cleavage within arrested ternary complexes.

RNA polymerase II may become arrested during transcript elongation, in which case the ternary complex remains intact but further RNA synthesis is blocked. To relieve arrest, the nascent transcript must be cleaved from the 3' end. RNAs of 7-17 nt are liberated and transcription continues from the newly exposed 3' end. Factor SII increases elongation efficiency by strongly stimulating the transcript cleavage reaction. We show here that arrest relief can also occur by the addition of pyrophosphate. This generates the same set of cleavage products as factor SII, but the fragments produced with pyrophosphate have 5'-triphosphate termini. Thus, the active site of RNA polymerase II, in the presence of pyrophosphate, appears to be capable of cleaving phosphodiester linkages as far as 17 nt upstream of the original site of polymerization, leaving the ternary complex intact and transcriptionally active.

RNA polymerase II promoter strength in vitro may be reduced by defects at initiation or promoter clearance.

We have measured the ability of three TATA box promoters, adenovirus 2 major late (Ad 2 ML), Ad 2 ML with a point mutation in the TATA box, and mouse beta-globin, to support abortive and productive RNA synthesis in vitro. We have also measured the ability of these promoters to direct the assembly of preinitiation complexes using a nuclease protection assay. The relative strengths in productive transcription, determined from the synthesis of RNAs 10 nucleotides or longer, were 12:6:1 for Ad 2 ML, mouse beta-globin, and the TATA mutant of Ad 2 ML. However, the TATA mutant was reduced only 4-fold in its ability to assemble preinitiation complexes, compared to Ad 2 ML. Complexes formed on the Ad 2 ML TATA mutant may therefore also be reduced in their ability to initiate transcription. The mouse beta-globin promoter directed assembly and initiation as well as Ad 2 ML, but the beta-globin transcription complexes were less able to clear the promoter, resulting in an increase in aborted transcripts at the expense of productive RNA synthesis. We have thus shown that the transcriptional strength of eukaryotic promoters may be determined not only at the step of transcription complex assembly but also at the level of promoter clearance and possibly at transcription initiation as well.

The lung-specific CC10 gene is regulated by transcription factors from the AP-1, octamer, and hepatocyte nuclear factor 3 families.

We have shown that a large fragment (-2339 to +57) from the rat CC10 gene directed lung-specific expression of a reporter construct in transgenic animals. Upon transfection, a smaller fragment (-165 to +57) supported reporter gene expression exclusively in the Clara cell-like NCI-H441 cell line, suggesting that a Clara cell-specific transcriptional element resided on this fragment (B. R. Stripp, P. L. Sawaya, D. S. Luse, K. A. Wikenheiser, S. E. Wert, J. A. Huffman, D. L. Lattier, G. Singh, S. L. Katyal, and J. A. Whitsett, J. Biol. Chem. 267:14703-14712, 1992). The interactions of nuclear proteins with a particular segment of the CC10 promoter which extends from 79 to 128 bp upstream of the CC10 transcription initiation site (CC10 region I) have now been studied. This sequence can stimulate both in vitro transcription in H441 nuclear extract and transient expression of reporter constructs in H441 cells. Electrophoretic mobility shift assays using extracts from H441, HeLa, rat liver, and fetal sheep lung cells were used to demonstrate that members of the AP-1, octamer, and HNF-3 families bind to CC10 region I. Transcription factors from H441 cells which are capable of binding to CC10 region I are either absent in HeLa, rat liver, and fetal sheep lung extracts or enriched in H441 extracts relative to extracts from non-Clara cells.

SII-facilitated transcript cleavage in RNA polymerase II complexes stalled early after initiation occurs in primarily dinucleotide increments.

RNA polymerase II ternary complex cleaves its nascent transcript in a 3'-->5' direction in the presence of elongation factor SII (Izban, M. G., and Luse, D. S. (1992) Genes & Dev. 6, 1342-1356; Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). We have characterized the cleavage products generated during the truncation process with a variety of stalled RNA polymerase II ternary complexes containing uniformly labeled transcripts. These complexes, which remain elongation competent, had stopped transcription because one nucleoside triphosphate was missing from the reaction mixture. Using a novel assay system, we demonstrate that cleavage occurs in predominantly dinucleotide increments, liberating 5'-phosphodinucleotides (pNpNs). In one instance with a particular C20 complex, the first cleavage event was equally partitioned between either a di-or trinucleotide increment with all subsequent truncations occurring by the preferred dinucleotide step. Our data indicate that both the kinetics and the exact increment of SII-facilitated transcript cleavage are influenced by transcript sequence.

The increment of SII-facilitated transcript cleavage varies dramatically between elongation competent and incompetent RNA polymerase II ternary complexes.

Elongation factor SII is required to increase the efficiency of transcription by RNA polymerase II through intrinsic arrest sites. RNA polymerase II ternary complexes exhibit a ribonuclease activity in the presence of SII, truncating nascent transcripts in a 3'-->5' direction. We show here that transcript cleavage is an obligatory step in re-establishing the elongation competency of complexes that have become blocked in elongation at an intrinsic arrest site. SII-facilitated transcript cleavage by these arrested complexes released 7-14 nucleotide RNA fragments. In contrast, SII-facilitated transcript cleavage by elongation competent complexes, which are stalled because of the absence of a nucleoside triphosphate from the reaction mixture, occurred primarily in dinucleotide increments. We can partially recreate the arrested phenotype and the preference for the large cleavage increment by stalling ternary complexes such that the 3'-end of the transcript contains consecutive U residues, which mimics the sequence of the 3'-ends of transcripts in arrested complexes.

cis-acting elements that confer lung epithelial cell expression of the CC10 gene.

To define cis-acting genetic elements responsible for cell-specific transcriptional regulation of the CC10 gene, DNA sequences spanning nucleotides -2338 to +49 of the rat CC10 gene were linked to a reporter gene coding for chloramphenicol acetyltransferase (CAT). In transient expression assays, CC10 sequences were capable of restricting CAT expression to a human lung adenocarcinoma cell line similar to pulmonary Clara cells. Transgenic mice harboring the hybrid RtCC10-CAT construct expressed high levels of CAT activity specifically within protein extracts of lung and trachea. Transcripts for the CAT reporter gene colocalized with those for the endogenous murine CC10 gene within the airways of transgenic mice. Functional analysis of deletion mutants identified stimulatory, inhibitory, and cell type-specific transcriptional regulatory elements. The results of gel retention and DNaseI protection assays suggest that a transcriptional stimulatory region located between -320 and -175, and a cell type-specific regulatory element located between -175 and +49, result from a series of protein-DNA interactions occurring at -220 to -205 and -128 to -86, respectively. Lung epithelial specific transcriptional regulatory elements described herein will be useful for expression of chimeric genes within epithelial cells lining the trachea, bronchi, and bronchioles of mice.

Factor-stimulated RNA polymerase II transcribes at physiological elongation rates on naked DNA but very poorly on chromatin templates.

We used an in vitro assay system based on HeLa cell core transcription components to examine transcript elongation by RNA polymerase II on either naked DNA or chromatin templates as a function of the three known elongation factors, IIS, TFIIF, and TFIIX. We demonstrate for the first time that mammalian RNA polymerase II can achieve physiological elongation rates on naked DNA templates in vitro. The addition of TFIIF alone gave this rate, although IIS was required to minimize the block to elongation at intrinsic termination sites. However, IIS and TFIIF provided only a slight increase in the very poor elongation efficiency of RNA polymerase II on chromatin templates. The addition of TFIIX to reactions containing IIS and TFIIF reduced the elongation rate on naked DNA templates but slightly increased the elongation efficiency on chromatin. The ability of elongation factors either separately or in combination to stimulate transcription on naked DNA and chromatin templates was also examined.

The RNA polymerase II ternary complex cleaves the nascent transcript in a 3'----5' direction in the presence of elongation factor SII.

The process by which RNA polymerase II elongates RNA chains remains poorly understood. Elongation factor SII is known to be required to maximize readthrough at intrinsic termination sites in vitro. We found that SII has the additional and unanticipated property of facilitating transcript cleavage by the ternary complex. We first noticed that the addition of SII caused a shortening of transcripts generated by RNA polymerase II at intrinsic termination sites during transcription reactions in which a single NTP was limiting. Truncation of the nascent transcript was subsequently observed using a series of ternary complexes artificially paused after the synthesis of 15-, 18-, 20-, 21-, and 35-nucleotide transcripts. Transcripts as short as 9 or 10 nucleotides were generated in 5-min reactions. All of these shortened RNAs remained in active ternary complexes because they could be chased quantitatively. Continuation of the truncation reaction produced RNAs as short as 4 nucleotides; however, once cleavage had proceeded to within 8 or 9 bases of the 5' end, the resulting transcription complexes could not elongate the RNAs with NTP addition. Transcript cleavage requires a divalent cation, appears to proceed primarily in 2-nucleotide increments, and is inhibited by alpha-amanitin. The catalytic site of RNA polymerase II is repositioned after transcript cleavage such that polymerization resumes at the proper location on the template strand. The extent and kinetics of the transcript truncation reaction are affected by both the position at which RNA polymerase is halted and the sequence of the transcript.

Abortive initiation is increased only for the weakest members of a set of down mutants of the adenovirus 2 major late promoter.

We have shown that accurate initiation of productive RNA synthesis in vitro at the adenovirus 2 major late promoter is accompanied by abortive initiation of very short transcripts (Luse, D. S., and Jacob, G. A. (1987) J. Biol. Chem. 262, 14990-14997). We made a set of sequence variants of this promoter, using every possible base at position -28 (in the TATA box) in the context of either the normal base (A) or a T at position +1 on the nontemplate strand. All changes from wild type reduced promoter strength. The two weakest promoters were 10- and 30-fold less active than wild type in productive RNA synthesis. We tested the possibility that the down mutations also caused an increase in the proportion of in vitro initiations which are abortive. This effect was seen only with the two weakest members of the promoter set. For these promoters, which share an A----C change at the -28 position of the TATA box, the ratio of abortive to productive initiations was 3-4-fold higher than for the other promoters. Interestingly, the sequence change at +1, although a down mutation, did not lead to an increase in abortive initiation.