From the
Production of full-length runoff transcripts in vitro and functional mRNA in vivo is sensitive to the drug
5,6-dichloro-1-
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)(
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-
Two
in vitro systems that best represent the features of the
in vivo situation are nuclear extracts from HeLa and
Drosophila Kc cells (K
Here we report the biochemical fractionation of K
First column fractionation of K
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 K
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.(
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 K
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.
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
)
(16) all
encounter blocks to elongation.
-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) .
N). 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 K
N 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) .
N 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.
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.
N 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.
N
yielded a single fraction capable of supporting efficient elongation in
the washed complex assay. Therefore, we undertook the fractionation of
K
N 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 K
N 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 K
N 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 K
N, but
produced only 10% of the DRB-sensitive transcripts (compare
K
N 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.
)
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.
N, 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.
-D-ribofuranosylbenzimidazole; P-TEF and
N-TEF, positive and negative transcription elongation factors,
respectively; FT, flow-through fraction.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.