INTRODUCTION

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Transcriptional elongation is a distinct and important step in the transfer of genetic information from gene to protein. Transcriptional elongation by Escherichia coli RNA polymerase is discontinuous in vitro (24, 28). The ternary elongation complex, consisting of RNA polymerase, DNA, and nascent RNA, can isomerize into an "arrested" conformation that is stable but incompetent for further elongation. Importantly, such isomerization depends on the transcribed DNA sequence. Yeast RNA polymerase II (Pol II) also arrests transcription in vitro (19, 26), suggesting that discontinuous elongation is a property of eukaryotic RNA polymerases. In addition, transcript cleavage factor TFIIS significantly contributes to the fidelity of Pol II transcription in vitro (17, 36), perhaps as a consequence of its antiarrest activity. However, as these in vitro experiments were performed under artificial conditions, they do not address whether transcriptional arrest can occur in vivo. If transcriptional arrest occurs in vivo, the stalled RNA polymerase would prohibit any further transcription, and DNA in the transcription bubble might be susceptible to mutagenesis. In addition, a stalled elongation complex might be recognized as a damage signal, and it has been suggested that defects in elongation are associated with recombination between direct repeats (5, 33).

Discontinuity of transcription elongation in vivo has been suggested by the observation of transcriptionally engaged Pol II at the promoter-proximal regions of various eukaryotic genes (3). In yeast cells, Pol II can stall at the 5' end of the lacZ gene (1, 5), and blocking Kin28-dependent phosphorylation of the C-terminal tail of Pol II does not preclude association of the preinitiation complex with promoters (20, 23). However, in all these situations Pol II is stalled at or near the promoter, suggesting that the transcriptional defect may occur at the level of promoter clearance and not at that of elongation per se. Furthermore, there is no evidence that the observed transcriptional defect depends on the DNA sequence around the block.

A number of proteins can affect eukaryotic transcriptional elongation in vitro (3, 38). The best-studied transcriptional elongation factor is TFIIS, which induces transcript cleavage in arrested elongation complexes, thereby permitting stalled RNA polymerase to proceed downstream (41). TFIIS is highly conserved among eukaryotes, and it has functional homologues in prokaryotes (22) and archaea (13). In yeast, TFIIS interacts genetically with several Pol II subunits (2, 14, 15, 25), components of the Spt4/Spt5 elongation complex (12, 40), the Cdc68 component of the FACT elongation complex (30), and the Swi/Snf nucleosome remodeling complex (7). Loss of TFIIS or mutations that interact genetically with TFIIS confer sensitivity to 6-azauracil (6-AU), a drug that inhibits GTP biosynthesis and hence decreases the concentration of nucleotides in vivo (8, 27). These observations have led to the hypothesis that TFIIS and 6-AU affect transcriptional elongation in vivo and that TFIIS is the physiologically relevant antiarrest factor. However, direct evidence for transcriptional arrest and for the role of TFIIS in transcription elongation in vivo is lacking.

Understanding of transcription elongation in vivo is significantly limited by the lack of a direct assay for this process. Here, we designed an artificial arrest (ARTAR) sequence, and we demonstrate that Pol II can be arrested in vivo at a position far from the promoter in a DNA sequence-dependent manner. TFIIS can counteract this arrest, thereby demonstrating its antiarrest activity in vivo. In addition, we show that the ARTAR sequence does not function under conditions of high transcriptional activation unless cells are exposed to conditions (6-AU or reduced temperature) that are presumed to affect elongation in vivo. Conversely, TFIIS affects gene expression under conditions of high, but not low, transcriptional activation. Our results provide physical evidence for the discontinuity of transcription elongation in vivo, and they suggest that the recovery from transcriptional arrest can be functionally distinguished from transcriptional elongation under conditions of high activation.

	MATERIALS AND METHODS

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Strains and DNAs. pDK5, a multicopy URA3 plasmid containing the lacZ structural gene expressed from the GAL1 promoter, is a derivative of pRY131 (42). It contains a KpnI-BglII cassette with two in-frame stop codons that is inserted between residues Pro⁶⁸⁶ and Gln⁶⁸⁷, a position located in a functionally dispensable interdomain (domain IV) loop of -galactosidase. The KpnI-BglII cassette was inserted into pRY131 by a "nick-repair" procedure that employs homologous recombination in vivo. Specifically, SacI-cleaved pRY131 and a 690-bp PCR product containing the KpnI-BglII cassette flanked by corresponding lacZ homology regions (obtained by sequential PCRs using the product of the first reaction as a primer for the second reaction) were introduced into yeast, and Ura⁺ transformants that were white in galactose-grown cells were selected. Double-stranded oligonucleotides carrying the full-length ARTAR sequence (UU*C) and a mutant derivative lacking the upstream-most segment (U*C) were obtained by PCR using appropriate oligonucleotides. Both the full-length and truncated versions of the ARTAR sequence were flanked by KpnI and BglII and cloned between the KpnI and BglII sites of pDK5. The resulting plasmids were partially digested with EcoRI to remove the 2µm origin of replication and then religated, thereby generating URA3 integration plasmids.

LacZ expression assays. For the -galactosidase assays, cells were grown overnight at 30°C (unless otherwise indicated) in medium containing Casamino Acids and lacking uracil in the presence of 2% raffinose. In many experiments, glucose was added to 0.1%, a concentration that does not affect glucose repression but significantly increases cell growth, the galactose induction rate, and steady-state -galactosidase levels. Upon reaching an optical density of approximately 1, galactose was added to the desired concentration and cells were incubated for an additional 4 h. -Galactosidase values represent the average of at least six independent colonies and are accurate to ±20%. lacZ and GAL1 RNA levels were determined by hybridization to oligonucleotide probes (located approximately 100 nucleotides downstream from the initiation site) and digestion with S1 nuclease as described previously (16).

Chromatin immunoprecipitation. Pol II occupancy was determined by chromatin immunoprecipitation assays, which were performed essentially as described previously (23). Cells (400 ml) were grown overnight in medium containing Casamino Acids without uracil in the presence of 2% of the appropriate carbon source (glucose, raffinose, or galactose) and then treated with 1% formaldehyde for 15 min. Chromatin from these cells was fragmented by sonication, and the resulting material was immunoprecipitated with anti-HA antibody bound to protein A-Sepharose beads. Quantitative PCR determinations were performed with five primer pairs covering various lacZ regions (see Fig. 5A) or with three primer pairs corresponding to the GAL1 locus (one centered at the transcriptional initiation site, the others centered at +321 and +800).

	RESULTS

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Design of an ARTAR site. Our initial goal was to determine whether transcriptional arrest occurs in vivo. Towards this end, we decided to engineer an ARTAR site. To develop a simple genetic assay that specifically measures transcriptional elongation (as distinct from promoter clearance), we inserted the ARTAR site at a promoter-distal position of the E. coli -galactosidase (lacZ) gene that is dispensable for enzymatic activity (4, 9).

View larger version (31K): [in this window] [in a new window]   FIG. 1.   Rationale for the artificial arresting DNA sequence design. The modules of the ARTAR site and three elongation complexes (gray boxes) occupying them are shown schematically. There are three similar arresting sections containing a spacer (SPC), a strong stabilizing hybrid (SH), a weak oligo(U) destabilizing hybrid (UH), and a strong stopping downstream bubble-flanking duplex (DBF). C-UH is an oligo(U) destabilizing hybrid that is modified by the clamp-forming element. The nascent transcript (dark grey line), DNA (open box in both duplex and RNA-DNA hybrid forms), active Pol II centers (triangles), and transcription bubble borders (brackets) are indicated. The upper panel shows three elongation complexes stalled by DBFs and prone to backtracking because of the weak UH and SPC. These complexes are shown to isomerize (backtrack) into the arrested conformations shown in the bottom panel that are stabilized by the SHs. The hairpin at the downstream ternary complex is predicted to form because of the C-UH structure.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   The nucleotide sequence of the ARTAR site. The names of the ARTAR elements correspond to those described in the legend to Fig. 1.

The ARTAR sequence can arrest transcription in vivo. As we wished to assay elongation-specific events in vivo, the ARTAR sequence was inserted into the interdomain (domain IV) loop of -galactosidase between Pro⁶⁸⁶ and Gln⁶⁸⁷. This loop has minimal impact on lacZ activity (4, 9), and it is located more than 2,000 nucleotides downstream from the promoter. Wild-type and ARTAR site-containing lacZ derivatives expressed from the GAL1 promoter were integrated into the URA3 locus of wild-type and TFIIS deletion strains. For each of the resulting strains, we measured -galactosidase activity in cells grown in glucose (repressed conditions; Fig. 3A), raffinose (noninducing conditions that permit low-level transcriptional activation; Fig. 3B), and galactose (inducing conditions; Fig. 3C).

View larger version (50K): [in this window] [in a new window]   FIG. 3.   Effect of the ARTAR site and TFIIS on lacZ expression. -Galactosidase levels in the indicated strains (artar designates the mutated U*C derivative) grown in medium containing 2% glucose (A), 2% raffinose (B), and 2% galactose (C). Note the differences in scales for -galactosidase levels in the various panels. (D) RNA levels in the indicated strains grown in 2% galactose medium.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Inverse effects of the ARTAR site and TFIIS as a function of galactose (gal) concentration. -Galactosidase levels in the indicated strains grown in medium containing 2% raffinose, 0.1% glucose, and the indicated concentrations of galactose. The scale for -galactosidase levels is exponential.

Pol II can arrest at the ARTAR sequence in vivo. Although the above experiments suggest that the ARTAR sequence can affect transcriptional elongation in vivo, conclusive evidence requires direct observation of Pol II arrested at the ARTAR sequence. Although the transcriptional run-on assay has been widely used for measuring Pol II occupancy throughout the gene in vivo (3), this assay is based on the assumption that elongation-competent RNA polymerase becomes active after nuclei isolated from cells are treated with sarcosyl. As it is impossible to assess the validity of this assumption for the case of the ARTAR sequence, we instead measured Pol II occupancy in vivo using chromatin immunoprecipitation. Such experiments were facilitated by the fact that all the yeast strains described above contain an HA epitope-tagged version of the Rpb3 subunit of Pol II.

View larger version (42K): [in this window] [in a new window]   FIG. 5.   Analysis of Pol II occupancy by chromatin immunoprecipitation. (A) Diagram showing the location of primer pairs for analysis of Pol II occupancy over the lacZ region. (B) Cross-linked chromatin from the indicated HA-Rpb3-containing strains grown in glucose (glu), raffinose (raf), or galactose (gal) medium immunoprecipitated with an HA-specific antibody and analyzed by quantitative PCR primers to the indicated regions.

Other phenotypes of the ARTAR sequence relevant to transcriptional elongation. We used three phenotypic assays to independently address the relevance of the ARTAR sequence and TFIIS to transcriptional elongation. First, we examined transcriptional elongation through the ARTAR sequence in the presence of 6-AU, a compound believed to inhibit elongation by reducing intracellular GTP and UTP levels (8, 27, 38). Interestingly, in galactose-grown strains containing TFIIS, 6-AU dramatically affects lacZ expression in an ARTAR-dependent manner (Fig. 6). As expected, the absence of TFIIS causes a dramatic reduction in lacZ expression, with the effect being somewhat more pronounced when the ARTAR site is present. Thus, 6-AU can enhance the effect of the ARTAR site in vivo, thereby strengthening their links to transcription elongation.

View larger version (25K): [in this window] [in a new window]   FIG. 6.   ARTAR and TFIIS dependence of lacZ expression in the presence of the indicated concentrations of 6-AU.

View larger version (17K): [in this window] [in a new window]   FIG. 7.   ARTAR and TFIIS dependence of the kinetics of lacZ expression after induction with 2 or 0.1% galactose.

View larger version (13K): [in this window] [in a new window]   FIG. 8.   ARTAR and TFIIS dependence of lacZ expression in cells grown at 16°C.

	DISCUSSION

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A specific assay for transcriptional elongation in vivo. Although there are various biochemical assays for transcriptional elongation, it has been difficult to specifically assay elongation in vivo. Nuclear run-on assays, which are often used to analyze elongation in vivo, are actually performed under artificial biochemical conditions, and extrapolation of such results to the physiological state requires a number of assumptions about the properties of Pol II. In yeast, elongation has been analyzed by measuring lacZ RNA (or protein) levels based on the observation that various mutations can decrease lacZ expression at a step after transcriptional activation (5, 6, 10, 32). However, it is unclear whether the specific effect on lacZ expression is due to transcriptional elongation per se or rather to promoter clearance or some other process. Lastly, 6-AU sensitivity has been used as a genetic assay for elongation, although the mechanistic basis for this is unclear (8, 27, 34, 35).

The ARTAR sequence can arrest transcription in vivo. Five lines of evidence indicate that the ARTAR sequence can, under appropriate conditions, affect transcription in vivo in a manner that is promoter distal and sequence dependent. First, in the absence of TFIIS, there is a striking ARTAR-dependent increase in Pol II occupancy just prior to the ARTAR sequence (Fig. 5; regions C and D). Importantly, Pol II occupancy 500 bp downstream from the ARTAR site (region E) is reduced at least fivefold in comparison to that observed at region C. These observations provide direct physical evidence for transcriptional arrest in vivo. Second, in wild-type cells grown under conditions of low activation (raffinose medium), there is a 10-fold reduction in lacZ expression that depends on the ARTAR sequence (Fig. 3). Third, under conditions of high activation (galactose medium), 6-AU dramatically affects lacZ expression in an ARTAR-dependent manner (Fig. 6). Fourth, the ARTAR sequence delays galactose-inducible lacZ expression (Fig. 7). Fifth, the ARTAR sequence has a strong effect on lacZ expression under conditions of high activation and low temperature (16°C) (Fig. 8). Although these ARTAR-dependent effects occur under specific experimental conditions, their existence provides conclusive evidence for the discontinuity of transcription elongation in vivo.

TFIIS can function as an antiarrest factor in vivo. Chromatin immunoprecipitation experiments conclusively demonstrate that TFIIS can relieve transcriptional arrest at the ARTAR site. Specifically, under conditions of low activation (raffinose or glucose) there is a sharp increase in Pol II occupancy just before the ARTAR site in cells lacking TFIIS but not in wild-type cells. Under low-activation conditions, we presume that only a single Pol II molecule transverses the gene at any given time, such that transcriptional arrest results in a traffic jam of Pol II molecules just before the ARTAR sequence. Such transcriptional arrest is clearly observed in TFIIS deletion cells but not in wild-type cells, therefore demonstrating that TFIIS can function as an antiarrest factor in vivo. To our knowledge, these results represent the first physical evidence for an elongation-specific function of TFIIS in vivo.

Effect of TFIIS on transcriptional elongation in vivo. In addition to its ability to mediate antiarrest at the ARTAR site, TFIIS is important for lacZ expression under conditions of strong Gal4-dependent activation. This decreased lacZ expression in the absence of TFIIS is presumably due to a transcriptional elongation defect, because the level of GAL1 mRNA is not significantly affected. By this assay, TFIIS behaves similarly to the Hpr1, Tho2, Mfp1, Thp2, and Thp1 proteins that have been implicated in transcriptional elongation (5, 6, 10, 32, 33). As such, our results provide additional evidence that TFIIS is important for transcriptional elongation in vivo. This presumed elongation activity of TFIIS on lacZ may or may not be similar to the activity that allows TFIIS to read through the ARTAR sequence (see below).

Transcriptional elongation and recovery from transcriptional arrest are distinguishable functions in vivo. It is generally believed that transcriptional elongation and the relief of transcriptional arrest are mechanistically related and that both processes involve TFIIS. However, our results indicate that the ARTAR sequence and TFIIS differently affect transcription in response to particular experimental conditions. At 30°C, ARTAR is important for lacZ expression under conditions of low activation (raffinose), whereas TFIIS is not. Conversely, TFIIS is important under conditions of high activation (galactose), whereas ARTAR is not. At 16°C, lacZ expression is more significantly affected by the ARTAR sequence and less significantly affected by TFIIS. These observations suggest that the ARTAR sequence imposes a transcriptional block that is distinct from the ones hindering transcriptional elongation at high activation levels. This distinction might involve different functions of TFIIS and/or might arise from kinetic effects related to which parameters of the overall transcriptional process are rate limiting under particular experimental conditions.