From the Molecular Biology Program, Sloan-Kettering
Institute, New York, New York 10021 and the § Department
of Microbiology and Immunology, Weill Medical College of Cornell
University, New York, New York 10021
Received for publication, November 18, 2002, and in revised form, December 6, 2002
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ABSTRACT |
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RNA polymerase II (pol II) is subject to an early
elongation delay induced by negative factors Spt5/Spt4 and NELF,
which is overcome by the positive factor P-TEFb (Cdk9/cyclin T), a
protein kinase that phosphorylates the pol II C-terminal domain (CTD) and the transcription elongation factor Spt5. Although the
rationale for this arrest and restart is unclear, recent studies
suggest a connection to mRNA capping, which is coupled to
transcription elongation via physical and functional interactions
between the cap-forming enzymes, the CTD-PO4, and
Spt5. Here we identify a novel interaction between fission yeast RNA
triphosphatase Pct1, the enzyme that initiates cap formation, and
Schizosaccharomyces pombe Cdk9. The C-terminal segment of
SpCdk9 comprises a Pct1-binding domain distinct from the N-terminal Cdk
domain. We show that the Cdk domain interacts with S. pombe
Pch1, a homolog of cyclin T, and that the purified recombinant
SpCdk9/Pch1 heterodimer can phosphorylate both the pol II CTD and the
C-terminal domain of S. pombe Spt5. We provide genetic
evidence that SpCdk9 and Pch1 are functional orthologs of the
Saccharomyces cerevisiae CTD kinase Bur1/Bur2, a putative
yeast P-TEFb. Mutations of the kinase active site and the regulatory
T-loop of SpCdk9 abolish its activity in vivo. Deleting the
C-terminal domain of SpCdk9 causes a severe growth defect. We suggest a
model whereby Spt5-induced arrest of early elongation ensures a
temporal window for recruitment of the capping enzymes, which in turn
attract Cdk9 to alleviate the arrest. This elongation checkpoint
may avoid wasteful rounds of transcription of uncapped
pre-mRNAs.
The 5'-cap is the defining structural feature of eukaryotic
mRNA. Consisting of m7G linked via an inverted 5'-5'
triphosphate bridge to the initiating nucleoside of the transcript, the
cap is formed by enzymatic modification of pre-mRNAs as they
are being synthesized by RNA polymerase II (pol
II).1 Capping entails three
reactions: (i) the 5'-triphosphate end of the nascent pre-mRNA is
hydrolyzed to a diphosphate by RNA triphosphatase, (ii) the diphosphate
RNA end is capped with GMP by RNA guanylyltransferase, and (iii) the
GpppN cap is methylated by RNA (guanine-N7) methyltransferase (1).
Targeting of cap formation to pre-mRNAs depends on
interactions of the capping enzymes with the phosphorylated C-terminal
domain (CTD) of the largest subunit of pol II (Ref. 2 and citations
therein). Recruitment of the capping apparatus to the elongation
complex requires the TFIIH-associated CTD kinase (Kin28 in yeast, Cdk7
in mammals), which phosphorylates Ser-5 of the CTD heptad repeat
YSPTSPS (3, 4).
Other protein-protein contacts may also be involved in coupling capping
to pol II transcription elongation. The pol II elongation factor Spt5
binds directly to the triphosphatase and guanylyltransferase components
of the mammalian and Schizosaccharomyces pombe capping apparatus (5, 6). The fission yeast S. pombe employs a
distinctive strategy of cap targeting whereby the triphosphatase (Pct1)
and guanylyltransferase (Pce1) enzymes of the capping apparatus are not
associated physically with each other (as they are in budding yeast and
metazoans), but instead bind independently to the phosphorylated pol II CTD and to the unphosphorylated C-terminal domain of
Spt5 (6, 7). The S. pombe Spt5 CTD consists of tandem
repeats of a nonapeptide motif of consensus sequence TPAWNSGSK
(6).
The HIV Tat protein binds to the triphosphatase and guanylyltransferase
domains of mammalian capping enzyme Mce1 and up-regulates both
activities (8). Spt5 and Tat cooperate to regulate HIV transcription
elongation. Spt5 and its binding partner Spt4 comprise the
transcription elongation factor DSIF (DRB sensitivity-inducing factor) (9, 10). DSIF binds to pol II and, in conjunction with NELF,
represses elongation at promoter-proximal positions (Ref. 11 and
citations therein). Escape from the elongation delay depends on P-TEFb
(positive transcription elongation factor b), a DRB-sensitive protein
kinase that phosphorylates both the pol II CTD and Spt5 (11-15).
P-TEFb consists of two subunits, Cdk9 and cyclin T1, and it binds to
Tat via cyclin T1 (Ref. 13 and citations therein). Expression of a
dominant negative version of Spt5 in human cells results in stimulation
of transcription from the HIV-LTR and other promoters, implying that
wild-type human Spt5 is a negative regulator of transcription (10).
Depletion of Cdk9 or cyclin T in Caenorhabditis elegans
embryos results in a general shutoff of pol II transcription (16).
Rescue of heat-shock transcription in Cdk9-depleted embryos by
co-depletion of Spt5 and Spt4 provides evidence for an inhibitory role
for Spt5/Spt4 during transcription elongation in vivo
(16).
A purpose of this regulatory circuit may be to ensure timely capping of
the nascent pre-mRNA before committing pol II to processive elongation (8). In the case of HIV, Spt5-induced arrest at promoter-proximal sites would maximize the opportunity for recruitment of Mce1 to the elongation complex, by a multiplicity of Mce1
interactions with Tat, Spt5, and/or pol II CTD-PO4. Recent
studies show that Tat directly stimulates the cotranscriptional capping
of nascent HIV pre-mRNA (17). An inference is that Tat serves a
dual role in promoting capping during the transcription arrest and in
activating P-TEFb to then override the elongation block.
It remains unclear whether and how the capping apparatus fits into the
Spt5/P-TEFb axis during cellular gene expression. Here, by studying the
macromolecular interactions of the S. pombe capping apparatus, we identify a S. pombe Cdk9 homolog as a binding
partner for the S. pombe RNA triphosphatase Pct1. A
C-terminal domain of S. pombe Cdk9, separate from the kinase
domain, suffices for binding to Pct1 in vivo and in
vitro. We identify Pch1 as the cyclin partner of SpCdk9 and show
genetically that SpCdk9/Pch1 are the functional orthologs of the
S. cerevisiae CTD kinase Bur1/Bur2, a putative fungal
counterpart of P-TEFb (18). Based on these findings, we invoke an
Spt5-induced elongation checkpoint, the purpose of which is to recruit
the capping enzymes, in which P-TEFb-mediated release from the
elongation block may be facilitated by contacts between P-TEFb and a
component of the capping apparatus.
Yeast 2-Hybrid Screen--
The screen was performed as described
previously using BD-Pce1 and BD-Pct1 as the bait (7). Plasmid DNA
recovered from the strains that tested positive for both
HIS3 and lacZ expression was used as the template
for PCR amplification of the S. pombe DNA insert with
flanking primers specific for the AD-fusion plasmid. The PCR products
were gel-purified and then sequenced. The AD plasmid clones were
recovered after transformation into Escherichia coli
DH5 SpCdk9 and Pch1 2-Hybrid Fusion Plasmids--
Gene fragments
encoding truncated versions of S. pombe Cdk9 were generated
by PCR amplification using sense primers that introduced a
NcoI site at the 5'-end of the truncated coding region and
an antisense primer that introduced an BamHI site
immediately downstream of the intended stop codon. The PCR products
were digested with NcoI and BamHI and then
inserted into the 2-hybrid BD fusion vector pAS2-1. Missense mutations
K65A, E83A, D184N, and T212A in the kinase domain were introduced into
the BD-SpCdk9-(1-385) plasmid. The full-length cDNA encoding
S. pombe cyclin Pch1 was amplified by PCR from a cDNA
library and inserted into the 2-hybrid AD fusion vector pGAD-GH. All of
the inserts were sequenced to ensure that the genes were fused in-frame
to AD or BD and that no unwanted coding changes had been introduced
during amplification and cloning.
Recombinant Proteins from Bacteria and Protein Affinity
Chromatography--
S. pombe RNA triphosphatase Pct1 was
produced in E. coli as an N-terminal
His10-tagged fusion and purified from soluble bacterial lysates by Ni-agarose chromatography as described previously (7, 19).
GST-Cdk9-(386-591), GST-Cdk9-(386-523), and GST-Bur1-(411-657) were
produced in bacteria by cloning the Cdk9-(386-591), Cdk9-(386-523), and Bur1-(411-657) open reading frames into the GST fusion vector pGEX-KG, transforming the expression plasmid into BL21(DE3), and inducing recombinant protein expression with
isopropyl-1-thio-
20 µg of purified GST, GST-Cdk9-(386-591) or GST-Bur1-(411-657) was
adsorbed to 50 µl of GSH-Sepharose beads during a 1-h incubation at
4 °C in 300 µl of binding buffer (50 mM Tris-HCl, pH
8.0, 50 mM NaCl, 5% glycerol, 1 mM
dithiothreitol, 0.03% Triton X-100). The beads were then washed twice
with 1 ml of binding buffer to remove unbound protein. Then the beads
were mixed with 5 µg of purified Pct1 for 1 h at 4 °C in 50 µl of binding buffer. The beads were then concentrated by
microcentrifugation, and the supernatant was withdrawn. The beads were
resuspended in 1 ml of binding buffer and subjected to two rounds of
concentration and washing. After the second wash, the bound proteins
were eluted with 50 µl of binding buffer containing 10 mM
glutathione. Aliquots (25 µl) of the input and the bead-bound
fractions were analyzed by SDS-PAGE.
Yeast Strains and Expression Plasmids--
The BUR1
and BUR2 genes were deleted in the S. cerevisiae
diploid strain W303 and replaced with a cassette specifying
kanamycin-resistance (20). Correct gene targeting was confirmed by
Southern blotting. The diploids were sporulated and tetrads dissected.
No viable kanamycin haploids were derived from the BUR1
bur1::kanR diploid, indicative of
BUR1 being an essential gene. Sporulation of the BUR1
bur1::kanR diploid that had been transformed
with plasmid p360-BUR1 (BUR1 URA3 CEN) yielded viable
bur1::kanR haploids that were
incapable of growth in the presence of 5-FOA, a drug that selects
against the BUR1 URA3 plasmid. Kanamycin-resistant haploids
were derived from the BUR2 bur2::kanR
diploid, which formed colonies on YPD agar at 30 °C, but not at
37 °C. Mata haploids of the bur1
p360-BUR1 (BUR1 URA3 CEN) contains the complete
BUR1 open reading frame plus 595 bp of genomic DNA upstream
of the translation start codon and 648-bp downstream of the stop codon.
p360-BUR2 (BUR2 URA3 CEN) contains the complete
BUR2 open reading frame plus 974 bp of genomic DNA upstream
of the start codon. pYX-SpCDK9 (CEN TRP1 SpCDK9) and
pYX-SpCDK9* (CEN ADE2 SpCDK9) contain the full-length
cDNA encoding SpCdk9 under the transcriptional control of the yeast
TPI1 promoter. cDNAs encoding missense mutants K65A, E83A, D184N, and T212A of full-length SpCdk9 were cloned into pYX132
(CEN TRP1) under the control of the TPI1
promoter. A truncated gene SpCDK Recombinant Baculoviruses--
Recombinant baculoviruses
encoding either untagged SpCdk9 or His6-tagged Pch1 were
generated using the BAC-TO-BAC Baculovirus Expression System
(Invitrogen) according the instructions of the vendor. The full-length
SpCDK9 gene was PCR-amplified using primers that introduced
BamHI sites at the start codon and immediately 3' of the
stop codon. The PCR product was digested with BamHI and then
inserted into the donor plasmid pFastBac1. The full-length PCH1 gene was amplified using primers that introduced
an NcoI site at the start codon and an EcoRI site
immediately 3' of the stop codon. The PCR product was digested with
NcoI and EcoRI and then inserted into donor
plasmid pFastBac-HTb so as to fuse Pch1 in-frame to an N-terminal His
tag flanked by a TEV protease cleavage site. All of the inserts were
sequenced to ensure that no unwanted coding changes had been introduced
during amplification and cloning.
Expression and Purification of the Recombinant SpCdk9/Pch1
Complex--
7.5 × 108 Sf9 insect cells were
plated (25 × 106 per 150-mm culture dish) and
co-infected with recombinant viruses expressing SpCdk9 or His-Pch1 at a
multiplicity of 5 for each virus. After incubation at 27 °C for
48 h, the cells were dislodged by gentle scraping with a rubber
policeman and harvested by centrifugation. The pellets were stored at
Protein Kinase Assay--
Reaction mixtures (30 µl) containing
10 mM Tris-HCl (pH 7.5), 75 mM NaCl, 1 mM dithiothreitol, 1 mM MgCl2, 50 µM [ Novel Interaction of Pct1 with a S. pombe Cdk9 Homolog--
A
2-hybrid screen of ~100,000 transformants for
triphosphatase-interacting proteins using a Gal4 DNA-binding domain
(BD)-Pct1 fusion as bait yielded 16 His+ isolates, two of
which contained plasmids encoding the Gal4 activation domain (AD) fused
in-frame to C-terminal fragments of a predicted 591-amino acid S. pombe polypeptide with extensive similarity to human and
Drosophila Cdk9 (Fig. 1).
Thus, we named this previously uncharacterized S. pombe gene
product SpCdk9. Two different AD-Cdk9 fusion clones were isolated in
the 2-hybrid screen: AD-Cdk9-(313-591) and AD-Cdk9-(330-591). The
His+ and lacZ+ phenotypes required
cotransformation with BD-Pct1 and AD-Cdk9-(313-591) plasmids and
neither fusion plasmid activated the HIS3 or lacZ reporter genes when cotransformed with the BD or AD vectors (Fig. 1A).
SpCdk9 was not isolated in a 2-hybrid screen for binding partners for
the S. pombe guanylyltransferase Pce1. This same screen did
uncover the positive interactions between Pce1, the pol II CTD, and
Spt5 (6, 7). Furthermore, a directed 2-hybrid interaction assay using
BD-Pce1 and AD-Cdk9-(313-591) plasmids failed to activate reporter
gene expression (not shown). We conclude that SpCdk9 interacts uniquely
with the triphosphatase component of the S. pombe mRNA
capping apparatus.
An Autonomous Pct1-binding Domain of SpCdk9--
SpCdk9 consists
of an N-terminal protein kinase domain and a C-terminal Pct1-binding
domain. The amino acid sequence of SpCdk9 is most closely related to
the sequences of the HsCdk9 (372 amino acids) and DmCdk9 (401 amino
acids) subunits of human and Drosophila P-TEFb (21) and to
the essential S. cerevisiae cyclin-dependent kinase Bur1 (657 amino acids), which phosphorylates the pol II CTD and
is the putative functional equivalent of Cdk9 in budding yeast (18).
SpCdk9 and Bur1 are 200-250 amino acids larger than the metazoan Cdk9
proteins; this difference is attributable to the presence of C-terminal
domains in the fungal Cdk9 proteins that have no counterpart in HsCdk9
and DmCdk9 (Fig. 1B). The fission yeast, budding yeast, and
metazoan Cdk9 proteins display sequence similarity throughout their
N-terminal kinase domains. (Residues conserved in all four proteins are
highlighted by dots in Fig. 1B).
The C-terminal polypeptides Cdk-(313-591) and Cdk-(330-591) that were
isolated in the 2-hybrid library screen contain short segments derived
from the protein kinase domain. We subsequently found that an
explicitly engineered shorter AD fusion, to Cdk9-(386-591), which
starts downstream of the Cdk domain, was just as active in the 2-hydrid
reporter assays as the constructs isolated originally (Fig.
1A). We conclude that the kinase domain does not contribute to the Pct1-binding site of SpCdk9, which resides entirely within the
C-terminal domain. By testing a series of deletions of SpCdk9 in the
2-hybrid interaction assay, we delineated an 82-amino acid segment
from residues 442 to 523 that sufficed for Pct1 binding (Fig. 1A).
Pct1 Binds Directly to the SpCdk9 Carboxyl Domain in Vitro--
We
used affinity chromatography to analyze the interaction of Pct1 with
SpCdk9 in vitro. A glutathione S-transferase
(GST)-Cdk9-(386-591) fusion protein (Fig.
2A, lane 4) was
immobilized on GSH-Sepharose beads, which were then mixed with purified
Pct1. The input Pct1 (lane 1) was analyzed by SDS-PAGE along
with the material that bound to the GSH resin and was subsequently
stripped off with glutathione. We found that the Pct1 protein bound to
the GSH beads containing the GST-Cdk9-(386-591) fusion (lane
3), but did not bind at all to GSH beads alone (lane 5)
or to GSH beads containing just GST (lane 2). The input Pct1
and glutathione eluate fractions were also assayed for triphosphatase
activity. Approximately 55% of the input activity was retained on the
GSH beads containing GST-Cdk9-(386-591) (Fig. 2B,
lanes 1 and 3). Control assays showed that the
GST-Cdk9-(386-591) fusion protein by itself had no triphosphatase activity (lane 4) and that there was negligible retention of
triphosphatase activity on the GSH beads (lane 5) or the GSH
beads containing GST (lane 2). Pct1 also bound specifically
to the shorter fusion GST-Cdk9-(386-523) (data not shown).
S. pombe Cdk9 Interacts with S. pombe Cyclin Pch1--
Metazoan
P-TEFb is composed of Cdk9 and cyclin T subunits (22). In considering a
candidate cyclin partner for SpCdk9, we focused on S. pombe
Pch1 (23), an essential 342-amino acid polypeptide that resembles
mammalian cyclin T (Fig. 3B).
We found that full-length Pch1 displayed a 2-hybrid interaction with
the isolated N-terminal kinase domain of SpCdk9 (Cdk9 S. pombe Cdk9 and Pch1 Are Functional Orthologs of S. cerevisiae
Bur1 and Bur2--
As an initial genetic test of the hypothesis that
SpCdk9/Pch1 are functionally related to P-TEFb, we asked whether
S. pombe Cdk9 and Pch1 can function in S. cerevisiae in lieu of the putative P-TEFb subunits Bur1 and Bur2.
We constructed yeast bur1
Additional experiments showed that expression of either Pch1 or SpCdk9
alone could not complement the bur2
Our conclusions are that: (i) SpCdk9/Pch1 is a functional ortholog of
Bur1/Bur2 and (ii) there is a species-specific interaction between the
Cdk and cyclin subunits. To consolidate the point that the SpCdk9/Pch1
functional interaction is specific, we tested whether a different
S. pombe cyclin, Mcs2, could substitute for Pch1 in the
yeast complementation assay. Mcs2 is an essential cyclin that partners
with a kinase subunit Mcs6 to form the fission yeast ortholog of the
CTD kinase component of TFIIH (25). We found that a yeast
CEN plasmid expressing Mcs2 was unable to complement bur1 Mutations of the Kinase Active Site and T-loop Abolish SpCdk9
Function in Vivo--
Initial studies of Pch1 isolated from S. pombe extracts showed that the cyclin was associated with a CTD
kinase activity (23); however, the identity of the catalytic subunit
was not established. Given that the Bur1/Bur2 complex is important for
transcription in S. cerevisiae and that it phosphorylates
the pol II CTD (18, 24), we can tentatively impute similar activities
to SpCdk9/Pch1. We addressed this issue genetically using budding yeast
as a surrogate model. We introduced mutations in residues Lys-65 and
Asp-184 of SpCdk9 that are predicted, based on crystal structure of the activated Cdk2-cylinA-substrate complex (26), to be constituents of the
kinase active site. (The residues that were mutated are shaded in Fig. 1B). We found that the K65A and
D184N mutations abolished SpCdk9's ability to complement
bur1
We also tested the effects of alanine substitution for Thr-212, a
conserved residue (Fig. 1B) corresponding to the regulatory phosphorylation site in the T-loop of Cdk2. The T212A mutation abolished SpCdk9 complementation of bur1 Mutation of Glu-83 of SpCdk9 Elicits a Severe Conditional Growth
Defect--
Glu-83 of SpCdk9 corresponds to the conserved glutamate of
the so-called PSTAIRE helix characteristic of Cdks. In the crystal structure of the activated Cdk2-cyclin A-substrate complex, the glutamate side chain forms an ion pair with the essential lysine (equivalent to Lys-65 of SpCdk9) that coordinates the Pct1 Binds to the C-terminal Domain of Bur1--
The Pct1-binding
domain of SpCdk9 is related structurally to the segment of S. cerevisiae Bur1 downstream of the kinase domain, to an extent of
62 positions of amino acid side chain identity or similarity within a
173-amino acid segment of SpCdk9 (Fig. 1B). The 82-amino
acid Pct1-binding module encompasses 28 positions of identity or
similarity. This raises the prospect that the interactions of Cdk9/Bur1
with RNA triphosphatase are conserved among fungi. Indeed, we found
that Pct1 bound specifically in vitro to a fusion protein
composed of GST linked to the C-terminal domain of Bur1-spanning amino
acids 411-657 (Fig. 2A). Measurement of bound
triphosphatase activity suggested that the affinity of Pch1 for the
Bur1 domain was comparable to its affinity for the SpCdk9
triphosphatase-binding domain (Fig. 2B). We also found that
S. cerevisiae RNA triphosphatase Cet1 (which is structurally
similar to Pct1) binds to the C-terminal domain of SpCdk9 (data not shown).
The C-terminal Domain Is Important for SpCdk9 Function in
Vivo--
Our identification of SpCdk9 as a binding partner for RNA
triphosphatase prompts the question: is the interaction between the two
proteins relevant in vivo? Having localized a
triphosphatase-binding element within the C-terminal domain of SpCdk9,
distinct from the kinase catalytic domain, we asked whether deletion of
the C-terminal domain impacts on SpCdk9 complementation of
bur1 Protein Kinase Activity of Recombinant
SpCdk9/Pch1--
Recombinant baculoviruses were engineered to express
native SpCdk9 or His-tagged Pch1 under the control of the viral
polyhedrin promoter. The tagged Pch1 allele was constructed so as to
place a TEV protease cleavage site at the junction between the short N-terminal His tag and the start of the Pch1 polypeptide sequence. Soluble lysates prepared from insect cells coinfected with the SpCdk9-
and His-Pch1-expresssing viruses were adsorbed to Ni-agarose to achieve
an affinity-purification of the His-tagged Pch1 and any associated
polypeptides (Fig. 7A). A
~43-kDa polypeptide corresponding to His-Pch1 was the predominant
species recovered by elution of the bound material with 150 mM imidazole. The initial identification of this species as
His-Pch1 was predicated on the following evidence: (i) treatment of the
peak imidazole eluate with TEV protease resulted in conversion of the
43-kDa polypeptide to a 41-kDa polypeptide, whereas the electrophoretic
mobility of the other polypeptides in the mixture was unaffected; and
(ii) Edman sequencing of the 41-kDa digestion product after transfer
from the SDS gel to a polyvinylidene difluoride membrane yielded the
N-terminal sequence Gly-Ala-Met-Gly-Glu-Val-Ile-Lys, which corresponds
precisely to the predicted sequence of the recombinant Pch1 protein
immediately flanking the TEV protease cleavage site. The imidazole
eluate also contained a prominent 68-kDa polypeptide, consistent with the predicted size for SpCdk9.
The peak imidazole eluate fraction was subjected to zonal velocity
sedimentation in a glycerol gradient. The sedimentation profile of the
component polypeptides was determined by SDS-PAGE (Fig. 7B).
The 43-kDa Pch1 polypeptide peaked in fractions 13-15, with a
prominent shoulder on the heavy side of the peak. The 68-kDa polypeptide also peaked in fractions 13-15, with a slight shoulder on
the heavy side. Attempts to sequence the 68- and 43-kDa polypeptides present in the peak glycerol gradient fraction by Edman chemistry were
unsuccessful, suggesting that their N-terminal amino groups were
blocked by covalent modification. This problem was circumvented by
tryptic digestion of the peptides in situ in an excised gel slice, followed by MALDI-TOF mass spectroscopy of the digestion products. The peptide fingerprinting analysis unambiguously identified the 68-kDa species as S. pombe Cdk9 and confirmed the
identification of the 43-kDa species as Pch1 (data not shown). The
finding that untagged SpCdk9 coeluted with His-tagged Pch1 during the
Ni-agarose step indicates that SpCdk9 and Pch1 form a heteromeric
complex in the absence of any other yeast proteins.
The peak glycerol gradient fraction containing SpCdk9 and Pch1 was
tested for protein kinase activity with a variety of potential phosphate acceptor protein substrates. Activity was gauged by transfer
of 32Pi from [
The kinase activity profile across the glycerol gradient was gauged
using GST-Spt5 as a substrate (Fig. 7C). We detected a single discrete peak of kinase activity centered at fractions 13-15,
coincident with the peak of the SpCdk9 and Pch1 polypeptides. An
apparent sedimentation coefficient of 6.0 S was calculated by
comparison to marker proteins (catalase, 11.2 S, 248 kDa; bovine serum
albumin, 4.4 S, 66 kDa; and cytochrome c, 1.9 S, 13.4 kDa) that were centrifuged in a parallel gradient. We surmise that the
active kinase is a heterodimer of the 68-kDa SpCdk9 and 43-kDa Pch1 polypeptides.
mRNA capping is coupled to transcription elongation via
physical interactions between the cap-forming enzymes, the
phosphorylated pol II CTD, and the elongation factor Spt5 (5, 6, 17, 27). Here we report that fission yeast RNA triphosphatase, the enzyme
that initiates cap formation, interacts with SpCdk9, an ortholog of the
Cdk9 subunit of metazoan P-TEFb. These findings provide a rationale for
the arrest and subsequent reactivation of pol II elongation at promoter
proximal sites (Fig. 8).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
.
-D-galactopyranoside. GST-Cdk9 and
GST-Bur1 fusion proteins were purified from a soluble bacterial extract
by adsorption to glutathione-Sepharose 4B resin and elution with buffer
containing 10 mM glutathione, 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10% glycerol, 0.05% Triton X-100.
The eluate was dialyzed against buffer containing 50 mM
Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM
dithiothreitol, 10% glycerol, 0.05% Triton X-100 and stored at
80 °C. Protein concentrations were determined by using the BioRad
dye reagent with bovine serum albumin as the standard.
and bur2
strains were used in the present study.
C encoding only the
N-terminal kinase domain (amino acids 1-385) was cloned into a
CEN ADE2 vector under the control of the TPI1
promoter. pYX-PCH1 (CEN ADE2 PCH1) and pYX-PCH1* (CEN TRP1 PCH1) contain the full-length cDNA encoding Pch1 driven
by the TP11 promoter.
80 °C. All subsequent procedures were performed at 4 °C. Thawed
cells were resuspended in 30 ml of lysis buffer (20 mM
Tris-HCl, pH 7.5, 125 mM NaCl, 2 mM
2-mercaptoethanol, 10% glycerol, 0.05% Nonidet P-40, complete
protease inhibitor mixture (Roche Molecular Biochemicals), 10 mM imidazole, and 0.25% Triton X-100). The suspension was
incubated on ice for 20 min and insoluble material was removed by
centrifugation for 45 min at 17,000 rpm in a Sorvall SS34 rotor. The
soluble extract was mixed with 1.5 ml of Ni-nitrilotriacetic
acid-agarose beads that had been equilibrated with lysis buffer. The
slurry was incubated for 2 h with gentle shaking. The beads were
collected by centrifugation, washed four times with 40 ml of 10 mM imidazole in buffer A (20 mM Tris-HCl, pH
7.5, 125 mM NaCl, 2 mM 2-mercaptoethanol, 10% glycerol, 0.05% Triton X-100), then poured into in a 10-ml column and
washed with 1.5 ml of 20 mM imidazole in buffer A. Bound
proteins were eluted with 150 mM imidazole in buffer A; 1.5 ml fractions were collected during the elution. The polypeptide
compositions of the column fractions were monitored by SDS-PAGE. An
aliquot (0.2 ml) of imidazole eluate fraction 2 (Fig. 7A)
was applied to a 4.8-ml 15-30% glycerol gradient containing 20 mM Tris-HCl (pH 7.5), 125 mM NaCl, 2 mM dithiothreitol, and 0.05% Triton X-100. The gradient
was centrifuged at 50,000 rpm for 15.5 h at 4 °C in a Beckman
SW50 rotor. Fractions (~0.2 ml) were collected from the bottom of the tube.
-32P]ATP, ~5 µg of phosphate
acceptor protein as specified, and enzyme were incubated for 15 min at
22 °C. The reactions were halted by adding SDS to a 1% final
concentration. The products were analyzed by electrophoresis through a
12% polyacrylamide gel containing 0.1% SDS. Phosphorylated
polypeptides were visualized by autoradiographic exposure of the dried
gel. The phosphate acceptor proteins were as follows: recombinant
GST-Spt5-(801-990) or purified untagged Spt5-(801-990) containing the
C-terminal nonapeptide repeat array of S. pombe Spt5
(6); recombinant GST-Pol2 containing the complete array of 52 heptapeptide repeats of human pol II (a gift from Karen Lee and Rob
Fisher); purified GST alone (6); or calf thymus histone H1 (purchased
from Roche Molecular Biochemicals).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Interaction of RNA triphosphatase with
Cdk9. A, the BD-Pct1 and AD-Cdk9-(313-591) fusion
plasmids and the BD and AD vectors without inserts were transformed
pairwise into S. cerevisiae Y190. Transformants containing
the indicated plasmid pairs were selected and streaked on S.D.(-Trp,
-Leu, -His, 3-AT) agar medium. The plates were photographed after
incubation for 5 days at 30 °C. Trp+ Leu+
isolates containing the indicated plasmid pairs were patched on
S.D.(-Trp, -Leu) agar medium. The cell patches were photographed after
incubation for 1 day at 30 °C (Growth). The cell patches
were then tested for -galactosidase activity using the colony-lift
filter assay (lacZ). B, the amino acid sequence
of the kinase domain of SpCdk9 is aligned to the sequences of
Drosophila Cdk9 (DmCdk9), human Cdk9 (HsCdk9), and S. cerevisiae Bur1 (ScBur1). Positions of side chain identity and
similarity in all four Cdk proteins are indicated by dots.
Conserved residues subjected to mutational analysis are
shaded and indicated by
. A portion of the C-terminal
Pct1-binding domain of SpCdk9 is aligned with the homologous C-terminal
segment of ScBur1; positions of identity/similarity are indicated by
dots. The region of SpCdk9 from residues 442-523 comprising
the Pct1 binding module is shaded.
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Fig. 2.
S. pombe Cdk9 binds Pct1 in
vitro. Panel A, purified Pct1 (5 µg) was
mixed with GSH beads containing immobilized GST (lane 2),
GST-Cdk9 (386-591) (lane 3), or GST-Bur1 (411-657)
(lane 6), or with GSH beads alone (lane 5).
Aliquots comprising 50% of the bead-bound eluate fractions
(lanes 2, 3, 5, and 6) were
analyzed by SDS-PAGE, along with 2.5 µg of the recombinant Pct1
(lane 1), GST-Cdk9-(386-591) (lane 4), and
GST-Bur1-(411-597) (lane 7) protein preparations. The
Coomassie Blue-stained gel is shown. Panel B, aliquots (1 µl) of the fractions analyzed in panel A, lanes
1-7, were assayed for triphosphatase activity (19). The extent of
32Pi release from [ 32P]ATP is
shown.
C), but did not
interact with the isolated C-terminal triphosphatase-binding domain of
SpCdk9 (Cdk9
N) (Fig. 3A). The 2-hybrid interaction was
stronger with the isolated kinase domain (scored as ++ based on
His+ colony size) than with the full-length BD-Cdk9 fusion
protein. S. pombe Pch1 also resembles the S. cerevisiae cyclin Bur2 (24), which together with the Cdk9-like
Bur1 kinase comprises the putative S. cerevisiae P-TEFb
ortholog (18). Thus, we envision that SpCdk9 and Pch1 are the
constituents of S. pombe P-TEFb.
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Fig. 3.
Interaction of SpCdk9 with cyclin Pch1.
A, AD-Pch1 and BD-Cdk9 (full-length), BD-Cdk9 N (amino
acids 386-591), BD-Cdk9
C (amino acids 1-385), or AD vector without
insert were transformed pairwise into S. cerevisiae Y190.
Transformants containing the indicated plasmid pairs were selected and
streaked on S.D.(-Trp, -Leu, -His, 3-AT) agar medium. The plates were
photographed after incubation for 5 days at 30 °C. Robust growth by
BD-Cdk9
C plus AD-Pch1 on selective medium was scored as ++.
BD-Cdk9
N plus Pch1, which grew no better than the BD-Cdk9
C plus
AD control, was scored as
. Intermediate level of growth by BD-Cdk9
plus AD-Pch1 was scored as + on the basis of His+ colony
size. B, the amino acid sequence of Pch1 is aligned to the
sequences human cyclin T1. Positions of side chain identity and
similarity are indicated by dots.
and bur2
strains
suitable for plasmid shuffle. The bur1
mutation was
lethal, while the bur2
strain was viable but slow-growing at 30 °C and unable to grow at 37 °C. The instructive findings were that, whereas expression of SpCdk9 or Pch1 alone could not complement bur1
, coexpression of SpCdk9 and Pch1 did
complement growth of the bur1
strain (Fig.
4A). In this experiment, the S. pombe genes were on single-copy CEN plasmids
marked with either TRP1 or ADE2. Complementation
of bur1
was effective whether a TRP1 SpCDK9
plasmid was cotransformed with an ADE2 PCH1 plasmid or a
TRP1 PCH1 vector was cotransformed with ADE2
SpCDK9 (Fig. 4A). SpCDK9 could not support
growth of bur1
cells when cotransformed with an extra
copy of BUR2 on a CEN plasmid driven by the
native BUR2 promoter (Fig. 4A) or by the
TPI1 promoter (not shown).
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Fig. 4.
SpCdk9 is a functional ortholog of
S. cerevisiae CTD kinase Bur1.
A, complementation of bur1 requires both
SpCDK9 and PCH1. The yeast bur1
p360-BUR1 strain was transformed with the indicated plasmids or plasmid
pairs (see "Experimental Procedures" for plasmid nomenclature) and
individual transformants were selected on appropriate drop-out media.
Colonies were patched on drop-out medium and then streaked on agar
plates containing 0.75 mg/ml 5-FOA. The plates were photographed after
incubation for 3 days at 30 °C. B, mutations of the
protein kinase active site abolish Cdk9 function in vivo.
bur1
p360-BUR1 was transformed with TRP1
plasmids containing the indicated alleles of SpCdk9 plus a ADE2
PCH1 plasmid. bur1
complementation was tested by
plasmid shuffle. Lethal mutations (scored as
) were those that failed
to support growth during selection on 5-FOA at either 23 or 30 °C.
E83A cells were viable, but displayed cs and ts growth defects (see the
legend to Fig. 6). The point mutations were introduced into
BD-Cdk9(
C) and tested for 2-hydrid interaction with Pch1 as
described in the legend to Fig. 3.
temperature-sensitive phenotype, whereas coexpression of Pch1 and SpCdk9 from CEN
plasmids did rescue bur2
growth at 37 °C (Fig.
5). Although the coexpression of the
S. cerevisiae kinase Bur1 with the S. pombe
cyclin Pch1 also rescued the bur2
ts phenotype (Fig. 5),
control experiments showed that the complementation was attributable
solely to the introduction of an additional copy of the BUR1
gene (not shown). This agrees with the earlier report that
BUR1 is a dosage suppressor of bur2
(24).
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Fig. 5.
Pch1 is a functional ortholog of S. cerevisiae cyclin Bur2. The yeast bur2
strain was transformed with the indicated plasmids or plasmid pairs and
individual transformants were selected on appropriate drop-out media.
Colonies were patched on drop-out medium and then streaked on YPD agar
plates. The plates were photographed after incubation for 2 days at 30 or 37 °C.
or rescue the bur2
ts phenotype when
cotransformed with a plasmid expressing SpCck9 (not shown). Thus,
SpCdk9 and Pch1 comprise a genuine Cdk/cyclin pair.
when coexpressed with Pch1 (Fig. 4B). The
inactivating mutations were then introduced individually into the
2-hybrid fusion protein BD-SpCdk9
C and tested for interaction
in vivo with AD-Pch1. Neither K65A nor D184N affected the 2-hybrid interaction, as gauged by
histidine prototrophy (Fig. 4B). Thus, the lethality of the
kinase active site mutations is likely not attributable to effects on
interaction of SpCdk9 with its cyclin partner. Rather, the results
suggest that SpCdk9 protein kinase activity is essential for its
in vivo function.
and did not
affect the interaction of the kinase domain with Pch1 in the 2-hybrid
assay (Fig. 4B). These results suggest that SpCdk9 function
may be regulated by phosphorylation of the T-loop, either by
autophosphorylation or phosphorylation by a separate activating kinase.
-phosphate of
ATP (26). We replaced Glu-83 of SpCdk9 with alanine and tested the
mutant enzyme for bur1
complementation when coexpressed
with Pch1. Although E83A supported the growth of
bur1
cells under FOA selection at 30 °C, the
SpCDK-E83A mutants displayed a severe temperature-sensitive
and cold-sensitive growth phenotype on rich medium, i.e.
they were unable to form colonies at either 23 or 37 °C, although
they were viable at 30 °C (Fig. 6).
E83A cells were also unable to form colonies on YPD agar at 18 °C
(not shown). We found that an E83A mutant of the 2-hybrid fusion
protein BD-SpCdk9
C retained its 2-hybrid interaction in
vivo with AD-Pch1. The strength of the E83A-Pch1 interaction was
++ at both 30 °C and 23 °C. Thus, we infer that the conditional
growth phenotype of the E83A cells was likely not caused by
a defect in the interaction of SpCdk9 with Pch1, but rather a
conditional defect in kinase activity resulting from loss of the ion
pair to the essential lysine that binds ATP.
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Fig. 6.
The C-terminal Pct1-binding domain is
important for SpCdk9 function in vivo. bur1
p360-BUR1 was transformed with plasmids containing the indicated
alleles of SpCDK9 plus a PCH1 plasmid.
5-FOA-resistant colonies were selected at 30 °C and then tested for
growth on YPD agar at 30, 23, and 37 °C.
. We found that expression of the Cdk9
C protein
(containing amino acids 1-385) supported growth of bur1
cells under FOA selection at 30 °C. However, the
C
mutant displayed a severe cold-sensitive phenotype when grown on rich
medium at 23 °C (Fig. 6) and 18 °C (not shown) and a partial
temperature-sensitive phenotype at 37 °C (Fig. 6). Even at
permissive temperature (30 °C), the
C mutant formed
smaller colonies than did "wild-type" SpCDK9 cells (Fig. 6). We surmise that the C-terminal domain containing the Pct1-binding site is important for SpCdk9 function in vivo. Consistent
with this idea, we found that the deletion variant SpCdk9 (1-523), which contains the Pct1-binding site, complemented the
bur1
mutation without eliciting cold-sensitive or
temperature-sensitive growth defects (not shown).
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Fig. 7.
Recombinant SpCdk9/Pch1. A,
Ni-agarose chromatography. Aliquots (30 µl) of the infected cell
lysate (L), flow-through (F), 20 mM
imidazole wash (W), and serial 150 mM eluate
fractions (1-5) were analyzed by SDS-PAGE. A Coomassie Blue-stained
gel is shown. The positions and sizes (in kDa) of marker proteins are
indicated on the left. B, glycerol gradient
sedimentation. Aliquots (20 µl) of odd numbered gradient fractions
were analyzed by SDS-PAGE (fraction 1 is the bottom of the gradient,
fraction 25 is the top). A Coomassie Blue-stained gel is shown. The
polypeptides corresponding to SpCdk9 and Pch1 are denoted by
arrows on the right. C, kinase
activity profile of the glycerol gradient. Reaction mixtures contained
5 µg of GST-Spt5-(801-990) and 3 µl of the indicated glycerol
gradient fractions. The reaction products were analyzed by SDS-PAGE,
and the signal intensity of the radiolabeled GST-Spt5 polypeptide was
measured by scanning the dried gel with a phosphorimager. The peaks of
marker proteins, catalase, bovine serum albumin (BSA), and
cytochrome c (cyt C), which were centrifuged in a
parallel gradient, are indicated by arrows. D,
kinase substrate specificity. Top panel, reaction mixtures
contained 3 µl of gradient fraction 14 and either no added
phosphate-acceptor protein (none) or 5 µg of
GST-Spt5-(801-990), untagged Spt5-(801-990), GST-Pol2, GST, or
histone H1. The reaction products were resolved by SDS-PAGE and
visualized by autoradiographic exposure of the dried gel. Bottom
panel, 5 µg of each phosphate acceptor protein were analyzed
separately by SDS-PAGE. A Coomassie Blue-stained gel is shown. The
positions and sizes (in kDa) of marker proteins are indicated on the
left.
-32P]ATP to the
acceptor protein to form a phosphoprotein adduct detectable by SDS-PAGE
and autoradiography (Fig. 7D). We readily detected
phosphoryl transfer to the C-terminal domain of S. pombe Spt5, which spans amino acids 801-990 and consists of 18 tandem repeats of a nonapeptide motif (consensus sequence TPAWNSGSK) (6).
Recombinant SpCdk9/Pch1 phosphorylated the nonamer array of Spt5 in the
context of a GST-Spt5 fusion protein or as tag-free Spt5-(801-990)
(Fig. 7D). No phosphoryl transfer was detected to GST alone.
The GST-Spt5 fusion protein was a better acceptor than free
Spt5-(801-990), perhaps because the GST-fused version is a dimer,
whereas the isolated Spt5 CTD is monomeric (6). SpCdk9/Pch1 also
phosphorylated the pol II CTD derived from mammalian pol II, consisting
of 52 tandem repeats of the CTD heptapeptide motif (Fig.
7D). In contrast, histone H1 was not a substrate for the
kinase. No labeled polypeptides were detected in the absence of an
exogenous acceptor.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Elongation checkpoint. The
experimentally documented binary interactions of S. pombe
capping enzymes Pce1 (guanylyltransferase) and Pct1 (triphosphatase)
with the pol II CTD-PO4, Spt5, and Cdk9 are indicated by
arrows. Binary interactions of S. pombe Cdk9 with
Pch1 (to form a putative counterpart of metazoan P-TEFb) and Spt5 with
Spt4 are documented herein and in Ref. 6. S. pombe Spt5/Spt4
is a putative homolog of metazoan DSIF, which acts in concert with NELF
to arrest elongation ( ). Like P-TEFb, S. pombe Cdk9/Pch1
phosphorylates the pol II CTD and Spt5 (indicated by arrows
flanked by PO4). The phosphorylation of the pol II CTD by
P-TEFb is implicated in reversing the elongation block.
Mammalian Spt5 in the context of DSIF interacts with pol II and arrests elongation. Spt5 also negatively regulates heat shock transcription in vivo in C. elegans (16). We presume that Spt5 in S. pombe has a similar function and we propose that the elongation arrest provides a kinetic window during which the capping enzymes Pct1 and Pce1 can be recruited to the elongation complex, via contacts with the Spt5 CTD nonamer array and also with the pol II CTD-PO4. We envision that cap formation occurs on the nascent chains within the arrested pol II complexes and that the presence of Pct1 on the pol II complex facilitates the recruitment of S. pombe P-TEFb, composed of SpCdk9 and its cyclin partner Pch1, via direct contacts between Pct1 and the C-terminal Pct1-binding domain of SpCdk9. By analogy with the mammalian system (13, 28), the proper positioning and/or activation of P-TEFb would trigger its phosphorylation of the pol II CTD and/or Spt5, thereby releasing the elongation complex from the arrest and committing pol II to processive elongation.
A key sensor in the S. pombe elongation checkpoint model illustrated in Fig. 8 is the presence of the capping enzyme Pct1 on the elongation complex, rather than presence of the cap structure. (Although we do not exclude a potential role for the cap, or a cap-associated protein, in elongation control, there is no evidence as yet to invoke such a model.) Pct1 provides a physical connection between Spt5 and SpCdk9. Studies in the mammalian system show that purified recombinant hSpt5 does not interact directly in vitro with recombinant Cdk9-cyclinT1 (P-TEFb) (14), implying the existence of a bridging component.
S. pombe Pct1 is the first instance in which an RNA
processing enzyme is physically linked to P-TEFb. The identification of the fungal P-TEFb homologs has been elusive, in part because of the
existence of multiple Cdk/cyclin pairs in S. cerevisiae that are capable of phosphorylating the pol II CTD (29). The studies of
Prelich and coworkers (18, 24) are persuasive in assigning the
essential Bur1/Bur2 kinase as the P-TEFb equivalent in budding yeast.
The fission yeast Cdk9 protein identified by us is structurally similar
to Bur1 and the similarity extends to the C-terminal Pct1-binding domain that is lacking in the metazoan Cdk9 proteins. Complementation of the S. cerevisiae bur1 and
bur2
mutants by coexpression of SpCdk9 and Pch1 provides
strong evidence that the fission yeast proteins are genuine orthologs
of Bur1/Bur2, while highlighting the species-specificity of the
Cdk-cyclin interactions. Analysis of the recombinant SpCdk9/Pch1
complex produced in baculovirus-infected insect cells shows that the
S. pombe proteins comprise a bona fide protein
kinase, with a putative heterodimeric quaternary structure. The
capacity of SpCdk9/Pch1 to phosphorylate the CTD arrays of both pol II
and Spt5 in vitro echoes the substrate specificity of
metazoan P-TEFb (14, 15). The fact that the in vivo function of SpCdk9 is abolished by mutations in Lys-65, which is predicted to
contact ATP, and Asp-184, which is predicted to coordinate magnesium in
the Mg-ATP complex, argues that protein phosphorylation is an essential
facet of SpCdk9's biological activity. The strong conditional
phenotypes elicited by the E83A mutation are in keeping with the
predicted indirect involvement of this residue in catalysis via its
positioning of the catalytic lysine. The finding that Thr-212 is
essential for SpCdk9 function suggests that SpCdk9 is subject to
allosteric activation via phosphorylation of the T-loop threonine. Yao
and Prelich (30) recently reported that Bur1 is phosphorylated on its
T-loop threonine by Cak1 and that T-loop phosphorylation stimulates the
CTD kinase activity of the Bur1/Bur2 complex. Their surprising finding
that an alanine mutation of the T-loop threonine of Bur1 did not affect
cell viability (30) contrasts with our result that the T-loop mutation
of SpCdk9 is lethal in yeast. It is conceivable that the SpCdk9/Pch1
complex is more acutely dependent than Bur1/Bur2 on a regulatory
phosphorylation event to attain the threshold level of kinase
activity required for growth of S. cerevisiae.
The C-terminal domain of SpCdk9 that includes the Pct1-binding site is
important for SpCdk9 function in vivo in bur1
complementation. Deletion of the C-terminal domain elicits a severe
conditional phenotype with both ts and cs growth
defects. The triphosphatase-binding domain of SpCdk9 is conserved in
S. cerevisiae Bur1. An equivalent of the C-terminal
triphosphatase-binding domain of fungal Cdk9 is lacking in metazoans,
but this makes perfect sense, insofar as the RNA triphosphatase
component of the metazoan capping apparatus is completely different in
its tertiary structure and catalytic mechanism from the RNA
triphosphatases of fungi (31, 32). Further analysis will be required to
test whether the growth defects of the SpCdk9
C mutation are
attributable, solely or partly, to the disruption of the interaction of
Cdk9/Pch1 with the capping apparatus.
Commitment of pol II to processive elongation without prior acquisition of a cap is potentially wasteful, given the role of the cap in facilitating splicing of the first intron and protecting the 5'-end from premature decay. Capping may also impact on proposed nuclear surveillance mechanisms for nonsense codons that rely on the translation machinery (Ref. 33 and citations therein). Recent studies in Drosophila suggest that ribosomes and translation factors associate with nascent pre-mRNAs within the nucleus prior to the removal of all introns and the acquisition of a poly(A) tail and that protein synthesis may occur at transcriptionally active nuclear regions (34). Cap-dependent recruitment of the translation apparatus to the nascent RNA is presumably a key event in ribosome-dependent nuclear mRNA surveillance.
We speculate that wasteful or defective rounds of transcription are
avoided by the imposition of an elongation checkpoint, whereby
Spt5/Spt4 plus other negative factors arrest the elongation complex
shortly after promoter clearance and reversal of the arrest by is
signaled by the presence of the capping apparatus on the elongation
complex (Fig. 8). This step is especially relevant to the control of
HIV gene expression, wherein the HIV Tat protein activates
cotranscriptional mRNA capping (17), and is likely to apply to
cellular gene expression, as evinced by the dual interactions of
S. pombe capping enzymes with Spt5 and the P-TEFb ortholog Cdk9/Pch1. Fleshing out the checkpoint model in the S. pombe
system will require many steps that are beyond the scope of the present study, including: (i) genetic dissection in S. pombe of each
of the putative checkpoint participants, (ii) purification and
biochemical characterization of the S. pombe Spt5/Spt4
complex, and (iii) the development of in vitro transcription
systems that display early elongation arrest and restart.
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ACKNOWLEDGEMENTS |
---|
We thank Karen Lee, Juliet Singer, and Rob Fisher for help with baculovirus construction and protein production in insect cells, and to Lynne Lacomis for protein microsequencing.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM52470.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Fax: 212-717-3623; E-mail: s-shuman@ski.mskcc.org.
Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M211713200
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ABBREVIATIONS |
---|
The abbreviations used are:
pol II, polymerase
II;
CTD, C-terminal domain;
AD, activation domain;
BD, binding domain;
GST, glutathione S-transferase;
P-TEFb, positive
transcription elongation factor b;
DRB, 5,6-dichloro-1--D-ribofuranosyl benzimidazole;
DSIF, DRB sensitivity-inducing factor;
HIV, human immunodeficiency
virus.
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