©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification of P-TEFb, a Transcription Factor Required for the Transition into Productive Elongation (*)

Nick F. Marshall , David H. Price (§)

From the (1) Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Production of full-length runoff transcripts in vitro and functional mRNA in vivo is sensitive to the drug 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB). We previously proposed the existence of an activity, P-TEF (positive transcription elongation factor) that functions in a DRB-sensitive manner to allow RNA polymerase II elongation complexes to efficiently synthesize long transcripts (Marshall, N. F. and Price, D. H.(1992) Mol. Cell. Biol. 12, 2078-2090). We have fractionated nuclear extracts of Drosophila melanogaster K cells and identified three activities, P-TEFa, factor 2, and P-TEFb, that are directly involved in reconstructing DRB-sensitive transcription. P-TEFb is essential for the production of DRB-sensitive long transcripts in vitro, while P-TEFa and factor 2 are stimulatory. P-TEFb activity is associated with a protein comprising two polypeptide subunits with apparent molecular masses of 124 and 43 kDa. Using a P-TEFb-dependent transcription system, we show that P-TEFb acts after initiation and is the limiting factor in the production of long runoff transcripts.


INTRODUCTION

The production of any functional eucaryotic mRNA requires efficient transcription elongation by RNA polymerase II. The choice to enter this productive elongation mode is not the default, but rather is a regulated step in gene expression (1, 2) . Experiments in human, murine, Drosophila, and Xenopus systems have demonstrated the existence of two classes of elongation complexes differing in their potential to produce full-length mRNA sized transcripts. Extensive studies of mammalian c-myc gene expression have shown that most initiation events only give rise to short RNA products (3, 4) . In cultured cells, a block to elongation at the end of the first exon regulates the levels of c-fos mRNA, in response to tumor promoters and intracellular calcium levels (5, 6, 7) . Significant blocks to elongation occur in the transcription of many other genes, including c-myb(8) , c-fms(9) , and adenosine deaminase (10, 11) . RNA polymerase II molecules are found arrested, during elongation, near the promoter on many genes in Drosophila melanogaster(12) . The transcription of viral messages from adenovirus (13) , simian virus 40 (SV40) (14) , minute virus of mice (15) , and human immunodeficiency virus (HIV)()(16) all encounter blocks to elongation.

The existence of two classes of transcription complexes differing in their elongation potential has also been demonstrated using the nucleoside analog 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB). The addition of DRB to mammalian cells in culture resulted in a 95% inhibition in the production of mature mRNA (17) . Nuclei isolated from cells pretreated with DRB have increased production of short, capped transcripts while labeling of longer RNAs is decreased (18, 19) . Similarly, the short transcripts generated from viral templates in cells infected with SV40 (20) and adenovirus (21) are enhanced while longer transcripts are suppressed with DRB treatment. DRB also inhibits production of long transcripts but leaves shorter products unaffected in injected Xenopus oocytes (4, 22) .

Two in vitro systems that best represent the features of the in vivo situation are nuclear extracts from HeLa and Drosophila Kc cells (KN). In HeLa nuclear extracts transcription initiating at the HIV long terminal repeat promoter gives rise to an abundance of transcripts terminated close to the promoter and only very limited amounts of longer transcripts. Addition of HIV Tat protein increases the number of DRB-sensitive elongation complexes that are able to make long transcripts (23) . Using Drosophila KN extracts only a fraction of RNA polymerase II molecules that initiate generate transcripts longer than several hundred nucleotides (24) . The addition of DRB to KcN extracts selectively inhibits the production of elongation complexes capable of sustained elongation. Two factors, P-TEF (positive transcription elongation factor) and N-TEF (negative transcription elongation factor), were proposed to control this behavior (25) .

Here we report the biochemical fractionation of KN and reconstitution of DRB-sensitive transcription in vitro. The reconstruction of DRB-sensitive transcription involves three chromatographically distinct protein fractions. Two of these activities are novel and have been termed P-TEFa and P-TEFb. The third activity, factor 2, was uncovered previously during the fractionation of KcN for factors necessary for the reconstruction of transcription initiation (26). P-TEFb has been purified to near homogeneity and is the only strictly required factor using the current, partially fractionated transcription system.


EXPERIMENTAL PROCEDURES

Materials

[-P]CTP (3000 Ci/mmol) was from ICN. Ribonucleoside triphosphates were from Pharmacia Biotech Inc. DRB (Sigma) was dissolved in ethanol to 10 mM and stored at -80 °C. Phosphocellulose (P-11) and DEAE-cellulose (DE-52) were obtained from Whatman and prepared according to Price et al. (26) . Phenyl-Sepharose (Pharmacia) was prepared according to manufacturer's instructions. All other chemicals were reagent grade.

Chromatography Methods and Initial Fractionation

Chromatography was carried out according to the principles outlined in Price et al. (26) . All columns were run in HGKEDP (25 mM HEPES, pH 7.6, 15% glycerol, 0-1 M KCl, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.1% of a saturated solution of phenylmethylsulfonyl fluoride in isopropanol) except for phenyl-Sepharose, which was run in HGAED (25 mM HEPES, pH 7.6, 15% glycerol, 0-1.5 M (NH)SO, 0.1 mM EDTA, 1 mM dithiothreitol). For both step and gradient elution protocols, columns were equilibrated and loaded at 0.1 M KCl in HGKEDP unless otherwise indicated. Where required, protein fractions were dialyzed to 0.1 M KCl before loading on the next column.

First column fractionation of KN on P-11 (see Fig. 1A) was according to a step protocol (26) . The 0.1 M KCl flow-through (FT) was brought to 0.3 M KCl and passed through DE-52 to remove the majority of nucleic acid content before further fractionation on an FPLC Mono Q column. The 0.1-0.3 M KCl step from P-11 was subjected to gradient fractionation on DE-52 and three separate activities, TFIIE, RNA polymerase II, and factor 2 (26) , were pooled and subjected to individual chromatography on Mono Q. The 0.3-0.4 M KCl step was concentrated 5-fold in 2-ml aliquots using Centricon-30 (Amicon) concentrators, subjected to centrifugation at 5,500 rpm (4,100 g) in a JA-17 rotor (Beckman) for 2-3.5 h at 4 °C, and dialyzed versus 75 mM HGKEDP until KCl concentration was 0.1 M or below. Samples of a 0.4-0.75 M KCl step from P-11 were brought to 0.15 M KCl and then passed through DE-52 column before further fractionation on an FPLC Mono S column.


Figure 1: Fractionation of KcN and reconstruction of DRB-sensitive transcription. A, summary of KcN fractionation detailed under ``Experimental Procedures'' Flatlines underneath column names indicate a step elution protocol, while angledlines indicate a gradient elution. Boldlines indicate the path to fractions required for DRB sensitivity. Thinnerlines indicate the path to fractions required for initiation. Elution points for factors not given previously (26) are indicated. B, dependence of DRB-sensitive transcription on the three indicated fractions. Fractions used in transcription reactions were generated using scheme shown in A. A ``+'' indicates the addition of an amount of factor determined as optimal for the generation of runoff when all three fractions were added together. Continuous labeling, transcription reactions are detailed under ``Experimental Procedures.'' Transcripts were analyzed in a 6% acrylamide-TBE-urea gel. Runoff indicates the position of the 520-nucleotide runoff transcript.



Purification of P-TEFb

The purification of P-TEFb was carried out twice from 70 to 90 ml of KcN and once from 155 ml of P-11 0.75 M step generated during the fractionation of Drosophila embryonic nuclear extract (gift from Will Zehring, Wayne State University). Although the exact chromatography steps differed during the three fractionations, the behavior of the P-TEFb activity from both sources was nearly identical. Two schemes used to purify the P-TEFb used in this work are summarized below.

P-TEFb used in Fig. 2A and 3B was purified from 90 ml of KN (approximately 3.0 g of protein). P-TEFb eluted between 0.55 and 0.65 M KCl during gradient elution of a 500-ml P-11 column from 0.15 M to 1.0 M KCl. P-TEFb-containing P-11 fractions (250 ml) were adjusted to 0.5 M (NH)SO, followed by loading onto a 26 ml phenyl-Sepharose column, which was eluted with a gradient from 0.5 M to 0 M (NH)SO. P-TEFb eluted from phenyl-Sepharose between 0.12 M and 0 M (NH)SO. P-TEFb-containing fractions (23 ml) at 0.06 M (NH)SO (equivalent to 150 mM KCl by conductivity) were pooled and allowed to flow through an 8-ml Mono Q equilibrated in 0.15 M KCl directly onto a 1-ml Mono S column. The Mono S column was then eluted with a gradient from 0.15 M to 0.45 M KCl during which P-TEFb eluted in 2.5 ml (50 µg of protein) between 0.25 M and 0.29 M KCl. A 200-µl sample from Mono S fraction 30 was loaded onto a 4.25 µl, 18%-35% glycerol gradient with a 500 µl of 1 M HGKE overlay and centrifuged at 55,000 rpm (287,000 g) in a Beckman SW 55 Ti rotor at 1 °C for 44 h.


Figure 2: Purification of P-TEFb. A and B, top, transcription gel analysis as in Fig. 1; bottom, silver-stained protein gel analysis of indicated fractions generated during the last two steps of the purification. -, no P-TEFb-containing fraction added; OP, column onput; runoff, 520-nucleotide runoff transcript. Proteins were analyzed in a 6-15% gradient SDS-polyacrylamide gel. Numbers to the left of gel indicate position of protein molecular size markers (10-kDa ladder from Life Technologies, Inc. or wide molecular weight standards, Sigma). Fraction33 is material collected from the bottom of glycerol gradient tube.



P-TEFb used in Fig. 2B was purified from 155 ml (265 mg protein) of P-11 0.4 M to 0.75 M step from embryonic nuclear extract. The initial P-11 step fraction was adjusted to 0.75 M (NH)SO before loading onto a 26-ml phenyl-Sepharose column, which was then gradient-eluted from 0.5 M to 0 M (NH)SO. P-TEFb activity eluted between 0.12 M and 0 M (NH)SO in 17 ml and was then dialyzed to 175 mM KCl before being passed through a 5.0-ml DE-52 column. The DE-52 flow-through (19 ml, 3.4 mg of protein) was loaded directly onto a 1-ml Mono S column, which was gradient-eluted from 175 mM to 500 mM KCl. P-TEFb activity eluting between 0.25 M and 0.29 M KCl was dialyzed and loaded onto a 1-ml Mono Q column at 50 mM KCl, followed by gradient elution from 50 mM to 450 mM KCl. P-TEFb activity was found in both the column flow-through and early gradient fractions. Both pools of P-TEFb activity were combined and rechromatographed over a 1-ml Mono S column loaded at 75 mM and step-eluted at 400 mM KCl. P-TEFb eluted in two 0.2-ml fractions. A 125-µl sample from one 0.2-ml fraction was loaded onto a 5-ml 15-35% glycerol gradient and centrifuged at 55,000 rpm (287,000 g) in a Beckman SW 55 Ti rotor at 1 °C for 40.5 h.

In Vitro Transcription

Two general types of transcription reactions were performed both using the actin Act5C template (24) linearized with HpaI. Pulse-chase reactions were generally as described previously (25) and began with a 10-min preincubation (6 µl/reaction) containing 20 mM HEPES, 5 mM MgCl, 45-50 mM KCl, 33 µg/ml DNA template, and extract or Kc-FT (see Fig. 3). Transcription was initiated by the addition of 2 µl of pulse solution, which contained 5 µCi of [-P]CTP and brought the reaction to 600 µM in GTP, ATP, and UTP. The true specific activity of the pulse is determined by the contamination of CTP in the other NTPs, which we have estimated to be 1 µM. The pulse was continued for 15 s after which the reaction was brought to 1.2 mM CTP by the addition of 12 µl of chase solution. After the indicated times, the reactions were stopped by the addition of 200 µl of a Sarkosyl solution (1% Sarkosyl, 100 mM NaCl, 100 mM Tris, pH 8, 10 mM EDTA, and 100 µg/ml tRNA). Sample workup and analysis of labeled transcripts in denaturing gels was as described previously (26) .


Figure 3: P-TEFb action after initiation. A, fractionation scheme for generation of Kc-FT. B, Pulse-chase transcription reactions (detailed under ``Experimental Procedures''). Amounts of KN (2 µl) and K-FT (4 µl) were balanced to give approximately equal levels of transcription initiation. P-TEFb was added to the reactions during the chase (1.0 P-TEFb is 1.0 µl of Mono S fraction 29). Transcripts were analyzed as in Figs. 1 and 2.



Transcription reactions to assay P-TEF activity in partially purified fractions were performed using a continuous labeling protocol, basically as described previously (26) . These reactions contained 20 mM HEPES, 5 mM MgCl, 600 µM each of GTP, ATP, and UTP, 30 µM CTP, 55-60 mM KCl, 3-4 µCi of [-P]CTP, 5 µg/ml DNA template, and various protein-containing fractions in a total volume of 12.5 µl. A typical P-TEFb assay contained 0.2 µl of DNase inhibitor, 0.2 µl of RNA polymerase II, 0.2 µl of dTFIIE, 1.5 µl of concentrated P-11 0.4 M step fraction, 0.1-0.2 µl of factor 2, and 0.5 µl of P-TEFa. A solution containing buffer, DNA, NTPs, and MgCl was added last to start the reactions. Reactions were incubated 20 min at 23 °C and stopped as described above.


RESULTS

We previously postulated the existence of a DRB-sensitive activity, P-TEF, that acted after initiation and allowed the generation of long runoff transcripts (25) . Although we suggested that it should be possible to purify P-TEF using an add-back assay to washed preinitiation complexes, no simple chromatography of KN yielded a single fraction capable of supporting efficient elongation in the washed complex assay. Therefore, we undertook the fractionation of KN and reconstruction of DRB-sensitive transcription using previous work on the chromatography of initiation factors as a guide (26). Fig. 1A shows the scheme that was used to fractionate KN with phosphocellulose (P-11) as the first column. Complete reconstruction after the first column using FT, 0.3 M, 0.4 M, and 0.75 M steps gives rise to DRB-sensitive transcription (data not shown). When these four fractions were subjected to further chromatography, it was possible to generate a system that only gave rise to DRB-insensitive transcripts (Fig. 1B, lefttwolanes). This reconstruction requires four fractions, DNase inhibitor, TFIIE, RNA polymerase II and a crude P-11 0.4 M step. The P-11 0.4 M step fraction includes at least TFIIB and TFIIF and probably TFIID and TFIIH.

DRB-sensitive Transcription Requires Three Protein Fractions

Besides the fractions required for efficient initiation, three additional fractions are required to reconstruct efficient DRB-sensitive transcription (Fig. 1B). Two of these fractions contain apparently novel activities, P-TEFa and P-TEFb, although P-TEFb may have been described previously as factor 6 (26) . The third is an activity described previously as factor 2 (26) . Using partially purified factors, P-TEFb appears to be the only fraction that is strictly required (Fig. 1B). P-TEFa and factor 2 both lead to stimulations in the level of runoff transcript, although other data lead us to believe that factor 2 may also be strictly required when using more purified fractions.() P-TEFb alone was able to support a very low, but detectable level of DRB-sensitive transcription (Fig. 1B). It is not clear if this low level of activity is due to the action of P-TEFb alone or because of contaminating amounts of either factor 2 or P-TEFa in the other fractions used.

P-TEFb Has Two Subunits

Since an absolute requirement for P-TEFb was evident, its purification was undertaken first. Details of the purification are found under ``Experimental Procedures.'' Analysis of the final two stages of the purification, chromatography on Mono S and glycerol gradient sedimentation, indicate that P-TEFb activity correlates with fractions containing two polypeptides with apparent molecular masses of 124 and 43 kDa (Fig. 2, A and B). P-TEFb was purified to near homogeneity three times, twice from KN and once from Drosophila embryonic nuclear extract, with nearly identical results. Fig. 2A shows the results from the fourth column in one of the KN purifications (see ``'' under ``Experimental Procedures''), while Fig. 2B is the last stage of purification from embryonic nuclear extract (see ``''). Glycerol gradient analysis of P-TEFb purified using gave nearly identical results (data not shown). Comparison of the sedimentation of known proteins to that of P-TEFb suggests that the factor is a heterodimer (data not shown).

P-TEFb Acts after Initiation

After purification of P-TEFb we realized that it should be easy to generate a transcription competent nuclear extract depleted of P-TEFb activity. As shown in the diagram in Fig. 3A, KN was passed through P-11 at 0.4 M KCl. The resulting K-FT was capable of initiating transcription as efficiently as whole KN, but produced only 10% of the DRB-sensitive transcripts (compare KN with K-FT alone lanes in Fig. 3B). Addition of P-TEFb to the K-FT restored the ability to generate DRB-sensitive runoff transcripts (Fig. 3B). The level of DRB-sensitive transcription achieved was dependent upon the concentration of P-TEFb added back (compare 1.0 and 2.5 P-TEFb lanes in Fig. 3B). Even with the highest levels of P-TEFb added back, the ratio of DRB-sensitive to abortive transcripts remained similar to that seen previously in extract (25) . Importantly, since P-TEFb was added to the reactions during the chase, the factor must have acted after initiation on the early elongation complexes.


DISCUSSION

We have identified three novel activities responsible for the regulation of transcription elongation. One of these activities, P-TEFb, was purified to near homogeneity, and its polypeptide components were identified. P-TEFb is an essential component of the P-TEF system, which regulates the production of long transcripts in vitro(25) . Its particular chromatographic properties and subunit composition distinguish it from any of the known basal initiation factors with the possible exception of TFIIJ (27) . At least one form of IIJ elutes from P-11 above 0.5 M KCl and the purified factor has two subunits of 33 and 95 kDa. A HeLa single-stranded DNA agarose column fraction that contains both IIA and IIJ did not substitute for P-TEFb.() The subunit composition and activity does not match that of any published Drosophila transcription factor. In particular, P-TEFb is unlike the elongation factors TFIIF (factor 5) or DmS-II because it does not stimulate the elongation rate of purified RNA polymerase on dC-tailed templates.() A slight stimulation of the elongation rate of RNA polymerase II in a dC-tailed template assay was seen with some P-TEFb-containing fractions, but the activity did not correlate with P-TEFb activity.() This activity was chromatographically separated from P-TEFb and could have been due to the presence of the Drosophila equivalent of S-III (28, 29) . Based on chromatographic properties, the factor identified by Chodosh et al.(30) in crude fractions from HeLa nuclear extract was not P-TEFb, but possibly the human equivalent of factor 2. Recently, Tat-SF was identified as a factor required for Tat activation of HIV transcription (31) . The chromatography of this protein on anion exchange resins is most similar to factor 2 or P-TEFa, not P-TEFb.

Although P-TEFb is not required for initiation, does not associate strongly with preinitiation complexes (25) , and has been shown here to act during elongation, it is not clear if there is a requirement for a specific type of initiation complex for P-TEFb to function. Although it was possible to equal and even surpass the level of DRB-sensitive transcripts achievable by KN, there appears to be an upper limit to the number of complexes that P-TEFb can act on from a fixed amount of K-FT (data not shown). Whether this constraint is time-based or actually represents a limitation in the number of P-TEFb affectable complexes is unclear. The timing of DRB sensitivity (25) and P-TEFb action appear to coincide closely. This is consistent with the activity of P-TEFb being inhibited by DRB. Since DRB is canonically a kinase inhibitor, this suggests that P-TEFb might be a kinase. Likely targets for a transcription factor kinase that acts early during elongation would be RNA polymerase II or a basal initiation factor such as TFIIF. The subunit composition of P-TEFb displays no obvious similarity to known kinases involved in transcription regulation, but detailed analysis of its activity and/or protein sequence will be required to confirm this.

Our current model is that P-TEFb recognizes some aspect of the early elongation complex and carries out its DRB-sensitive function on this complex. P-TEFb may remain associated with the complex or it may be released, but the complex is DRB-resistant after that point (24) . The roles of factor 2 and P-TEFa in this process remain to be elucidated. Neither P-TEFb alone nor the combination of P-TEFa, P-TEFb, and factor 2 could support efficient DRB-sensitive elongation when added back to washed elongation complexes (data not shown). This may have been due to lack of TFIIF or S-II type elongation factors or possibly due to the absence of some other unidentified factor. Further work in this area will be concentrated upon defining the mechanism of action of P-TEFb including potential targets for its action. The purification and characterization of both factor 2 and P-TEFa are currently under way.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant R01-GM35500. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 319-335-7910; Fax: 319-335-9570.

The abbreviations used are: HIV, human immunodeficiency virus; DRB, 5,6-dichloro-1--D-ribofuranosylbenzimidazole; P-TEF and N-TEF, positive and negative transcription elongation factors, respectively; FT, flow-through fraction.

Z. Xie and D. H. Price, unpublished data.

L. Zawel, D. Reinberg, N. F. Marshall, and D. H. Price, unpublished data.

D. R. Chafin and D. H. Price, unpublished data.

M. Doro and D. H. Price, unpublished data.


ACKNOWLEDGEMENTS

The factor 2 fraction was provided by Zhi Xie. We also thank Will Zehring for giving us the high salt P-11 fraction from embryonic nuclear extract.


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