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INTRODUCTION |
Over the last few years a wealth of biochemical
and genetic data have revealed extended coupling between nuclear events
of gene expression (1, 2). In particular, transcription by RNA
polymerase II (pol II)1 is
coupled to mRNA processing, including 5' capping and splicing and
3'-end formation of the transcript. This coupling is achieved by the
binding of mRNA processing factors to the phosphorylated C-terminal repeat domain (CTD), a mobile extension of the
catalytic core of pol II (3). The CTD becomes phosphorylated during
transcription initiation and remains phosphorylated during RNA chain
elongation. Several CTD kinases have been described (4), but only one
CTD phosphatase, called Fcp1, is known (5, 6). In addition to pol II,
several other proteins of the transcription machinery are
phosphorylated (7), and RNA processing complexes contain phosphoproteins as well. Phosphorylation and dephosphorylation events
are thus crucial for the regulation of transcription-coupled mRNA processing.
Ssu72 was originally identified in a yeast genetic screen. A mutation
in the ssu72 gene enhances a defect in the general
transcription factor IIB (TFIIB) that confers a shift in the
transcription start site (8). Indeed, Ssu72 binds directly to TFIIB
(9), and it also interacts with pol II, both genetically and physically (10, 11). Yeast Ssu72 is a subunit of the cleavage and polyadenylation factor (CPF), which together with cleavage factor I and poly(A) polymerase is sufficient for mRNA 3'-end formation (11, 12). Because RNA 3'-end formation and transcription termination are interlinked, and because Ssu72 functions also during transcription initiation, Ssu72 may be involved in a possible coupling between transcription termination and initiation (1). Recent data show that Ssu72 is also involved in transcription elongation (11). The Ssu72
mutation can increase pol II pausing and can counteract the toxicity of
6-azauracil, an inhibitor of pol II elongation (11). Yeast Ssu72 is
essential for viability (8) and shares 44% identical amino acid
residues with its human homologue. The high degree of conservation
suggests that Ssu72 has a similar function in all eukaryotic cells.
Thus Ssu72 is an essential and highly conserved protein involved in
eukaryotic mRNA biogenesis. However, the biochemical function of
Ssu72 remained unknown. Here we provide evidence that Ssu72 is a
phosphatase that resembles protein tyrosine phosphatases (PTPases).
PTPases can be divided in four families, including receptor-like,
intracellular, dual specificity, and low molecular weight (low
Mr) PTPases (13, 14). We demonstrate that Ssu72 shows similarities to the low Mr PTPase family
but that it also has distinguishing features. Possible functions of
this novel enzymatic activity during mRNA biogenesis are discussed.
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EXPERIMENTAL PROCEDURES |
Cloning and Site-directed Mutagenesis--
Gene ssu72
encoding for the 194-amino acid residue human isoform 1 was amplified
by PCR from human muscle cDNA using the oligonucleotide primers
5'-CGCATGCCATGGCAATGCCGTCGTCCCCGCTGCGGG-3' and 5'-GCTTTTCCTGCGGCCGCGTAGAAGCAGACGGTGTGC-3' (mutated
positions in bold, restriction sites underlined). Thereby, an
NcoI restriction site and codons for two additional amino
acids, methionine and alanine, were added to the 5'-end of the
gene, and at the 3'-end of the gene, the stop codon was removed and a
NotI site was added. PCR products were digested and ligated
into pET28b (Novagen), resulting in a construct that encodes for the
Ssu72 polypeptide chain plus a C-terminal hexahistidine tag. After
transformation of ligation products into Escherichia coli
XL1 blue cells, plasmid DNA was isolated, and DNA from positive clones
was sequenced. The resulting DNA sequence of gene ssu72
agrees with the published sequence (10) but deviates from the data base
entry for PNAS-120 (NCBI accession number AAK07538) at two amino acid
positions. Apparent mutations encode for residues Lys-182 and
Ser-183 instead of Arg-182 and Val-183, respectively. Point mutations
were introduced by site-directed mutagenesis with the two step PCR
overlap extension method. The mutated gene ssu72 C12S was
amplified from the cloned ssu72 plasmid DNA in two steps. In
the first step, two independent PCR reactions were carried out. The
first reaction was carried out with a T7 promoter forward primer and
the primer
5'-GCTCCGGTTCTGGTTGCTCGAGCTCACCACCGCCACCCG-3'. In the second reaction we used the reverse complement counterpart to
the latter primer and a T7 terminator reverse primer. In the second
step, the entire mutated gene was amplified with both T7 primers from
the PCR products of the first step. The resulting PCR product was
digested with NcoI and NotI and ligated into
pET28b (Novagen). For amplification of mutated genes ssu72 R18K,
D140A, and D143A, the primers
5'-GTTGTGCGCCTCCATGCTCTTGTTCTGGTTGCTCGAGCAC-3', 5'-CCTGGATGGCCACATTGACCACGTGC-3', and
5'-GCCTCCTCGTGGTTCGCCTGGATGTCCAC-3' were instead
used in the first step, respectively, together with their reverse
complement counterparts.
Protein Expression and Purification--
Plasmid DNA harboring
the human gene ssu72 was transformed into E. coli
BL21 DE3 CodonPlus RIL cells (Stratagene). Cells were grown at 37 °C
in LB medium supplemented with chloramphenicol and
kanamycin, both at concentrations of 50 µg/ml. Once the cell culture reached an A600 of 0.5, temperature was reduced to 20 °C, and cells were induced with 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside and grown
over night. Cells were harvested by centrifugation and suspended in
buffer A (50 mM Tris-Cl, pH 8.0, 500 mM NaCl,
10 mM
-mercaptoethanol), flash-frozen, and stored at
80 °C. All protein variants were purified as follows. Cell walls
were broken with a French press followed by DNase I (Roche Applied
Science) incubation on ice for 20 min. The slurry was cleared by
centrifugation, and the supernatant was loaded onto a
nickel-nitrilotriacetic acid-agarose column (Qiagen). The column was
washed with 20 column volumes (CV) of buffer A followed by 5 CV of
buffer B (buffer A but with 300 mM NaCl). Proteins were
eluted with buffer B containing in addition 250 mM
imidazole. Eluted proteins were diluted with 8 volumes of buffer C (50 mM Tris-Cl, pH 8.0, 1 mM EDTA, 1 mM DTT) and bound to a MonoQ column (Amersham Biosciences). The column was
washed with 2 CV of buffer C, and the protein was eluted in a gradient
of 25 CV from 0 to 1 M NaCl in buffer C. Peak fractions were applied to a Superose 12 HR gel filtration column (Amersham Biosciences), equilibrated with 50 mM Tris-Cl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 1 mM DTT.
Protein purity was judged by SDS-PAGE. Recombinant Ssu72 migrated on
SDS-PAGE with an apparent molecular mass of 30 kDa compared with
a theoretical molecular mass of 24.0 kDa. In size exclusion
chromatography, Ssu72 migrated like a globular protein of around 50 kDa
molecular mass, suggesting that Ssu72 forms a stable homodimer. Protein
concentration was determined with the Bradford assay (Bio-Rad). Under
near physiological buffer conditions, Ssu72 was highly soluble and
could be concentrated to 15 mg/ml. Pure protein samples were
flash-frozen in liquid nitrogen and stored at
80 °C.
Phosphatase Assays--
Cleavage of
p-nitrophenylphosphate (pNPP) was used to characterize the
enzymatic activity of Ssu72. Formation of the
p-nitrophenylate product was followed in real time by
measuring absorbance at 405 nm (
405 = 18 mM
1cm
1) with an enzyme-linked
immunosorbent assay reader. The pH optimum for pNPP cleavage was
determined in steps of 0.5 pH units by measuring product absorbance
after cleavage of 5 mM pNPP by 0.9 µM Ssu72 after a 15-min incubation at 37 °C. For assays between pH 4.5 and
7.5, 40 mM sodium citrate-HCl buffer was used. Between pH 7.5 and 9.0, 40 mM Tris-Cl buffer was used. For
determination of the temperature dependence of pNPP cleavage in the
range of 25 to 50 °C, product formation was monitored in steps of
5° after a 20-min incubation of 5 mM pNPP and 0.9 µM Ssu72 in 40 mM sodium citrate-HCl buffer,
pH 6.5. For reactions that included divalent metal ions, the buffer was
replaced by 50 mM Bis-Tris-HCl, pH 6.5, to circumvent the
chelating effect of citrate, and samples were incubated for 20 min. For
all inhibitor studies, product formation after a 20-min incubation was
measured for a substrate concentration of 5 mM. The
inhibitory anions [BeF3]
and
[AlF4]
were generated in situ by
the addition of 1 mM NaF and 100 µM BeCl2 or AlCl3, respectively. The reaction was
stopped by the addition of 9 volumes of 1 M
Na2CO3. Michaelis-Menten kinetic parameters
were determined by measuring initial reaction rates at various pNPP
concentrations in 50 mM citrate-HCl, pH 6.5, 10 mM EDTA, 1 mM DTT, and 0.9 µM
Ssu72. Data were fitted to Lineweaver-Burk equations with the program
Origin. The inhibitory constant for phosphate was determined by
measuring Km values at various inhibitor
concentrations followed by graphical evaluation.
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RESULTS |
Ssu72 Contains the Signature Motif of Protein Tyrosine
Phosphatases--
By visual inspection of the Ssu72 amino acid
sequence, we identified the signature motif
11VCX5RS19, which is
typical for PTPases. This motif comprises a cysteine and an
arginine residue that form part of the active site in all PTPases and
are involved in catalysis (15). In PTPases, the cysteine residue is
generally responsible for the nucleophilic attack of the substrate
phosphorus atom, leading to the formation of a phosphoenzyme
intermediate, whereas the arginine residue is involved in stabilization
of the transition state (16). Except for the signature motif, there is
no apparent sequence homology between Ssu72 and PTPases.
Ssu72 Cleaves a Phosphotyrosine Analogue--
To test Ssu72 for a
potential phosphatase activity, we cloned human Ssu72, over-expressed
the protein in E. coli, and purified it to apparent
homogeneity (see "Experimental Procedures"). We then subjected the
purified recombinant protein to a colorimetric assay based on cleavage
of the phosphotyrosine analogue pNPP (see "Experimental
Procedures"). Highly purified recombinant Ssu72 could indeed cleave
pNPP. Ssu72 activity was strongly dependent on the pH of the buffer and
was highest at pH 6.5. At pH 6.0, the protein showed 15% of its
maximum activity, and below pH 5.5, protein precipitation essentially
abolished activity. From pH 6.5 to 9.0, activity progressively
decreased to about 10% of the maximum. The temperature optimum for
Ssu72 activity was reached at 40 °C, but the protein showed 86% of
its maximum activity at 37 °C. To minimize experimental errors and
to avoid long incubation times that could lead to enzyme inactivation,
further experiments were performed at pH 6.5 and 37 °C
under near physiological conditions. Michaelis-Menten kinetics
and Lineweaver-Burk analysis revealed a Km value for
pNPP of 3.6 mM (Fig. 1),
comparable with values reported for the dual specificity phosphatase
VHR (17) and the human low Mr PTPase
HCPTB (18) but 1-2 orders of magnitude higher than that of two other
low Mr PTPase (Table
I).

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Fig. 1.
Phosphatase activity of Ssu72.
Lineweaver-Burk plots for the determination of Km
values for cleavage of pNPP by recombinant Ssu72 wild type
(A), Ssu72 variant D140A (B), and Ssu72 variant
D143A (C). Note the different scales used in
A, B, and C. The Km
values obtained are listed in Table I.
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Ssu72 Activity Is Impaired by Phosphatase Inhibitors--
We next
tested whether known phosphatase-inhibiting agents had an effect on
Ssu72 activity (Fig. 2A). The
anion [BeF3]
, a potent inhibitor of
phosphatases that form phosphoaspartate intermediates (19, 20), did not
show an effect on Ssu72 activity. Similar results were obtained with
the anion [AlF4]
. In contrast, vanadate
ions strongly inhibited Ssu72 activity. The addition of 1 mM orthovanadate reduced product formation below 5%.
Phosphate ions also inhibited Ssu72 activity but not as strongly as
vanadate. Michaelis-Menten analysis revealed that phosphate acts as a
competitive inhibitor (Fig. 2B). The Ki
value for phosphate, 4.3 mM, is close to the
Km value for pNPP. This shows that phosphate
efficiently competes with pNPP for binding to the active site,
suggesting that Ssu72 affinity for pNPP is governed by the binding of
the phosphoryl group.

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Fig. 2.
Inhibition of Ssu72 activity.
A, effects of EDTA (50 mM), metal ions (1 mM, light gray; 10 mM, dark
gray), phosphate (5 mM), orthovanadate (1 mM), [BeF3] , and
[AlF4] on Ssu72 activity (compare
"Experimental Procedures"). Data are shown as the percentage of
Ssu72 activity in the absence of inhibitors. B,
determination of the inhibitory constant Ki for
phosphate ions. Slopes of four individual Lineweaver-Burk plots from
Km determinations at various phosphate
concentrations were plotted against the four different phosphate
concentrations used. The slope of the resulting plot corresponds
to (Km/Vmax) + ([I]·Km/Ki·Vmax),
where [I] is the phosphate inhibitor concentration. A linear
regression for the values provides Ki as the
intersection with the x axis, where the slope is zero.
C, reversible oxidation and inactivation of Ssu72. Ssu72 was
oxidized by adding 0.01 or 0.001% H2O2 to 5 mM pNPP and 0.9 µM Ssu72 in 50 mM
citrate-HCl buffer, pH 6.5. After a 30-min incubation, DTT was added to
reduce Ssu72 and restore catalytic activity.
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The influence of divalent metal ions on Ssu72 activity was also
examined (Fig. 2A). The addition of 1 mM
Mg2+, Mn2+, or Ca2+ had essentially
no effect, but at 10 mM concentrations, product formation
was reduced to 60, 45, and 80%, respectively. In contrast, 1 mM Co2+, Ni2+, or Cu2+
were sufficient to abolish activity. Consistently, Ssu72 was inactive
after elution from a Ni-NTA-agarose column, but activity was rescued
after removal of bound Ni2+ ions with a MonoQ column. In
keeping with the negative effect of metal ions, EDTA enhanced Ssu72
activity slightly, most likely because of chelation of metal ion
traces. Consequently, 10 mM EDTA was included in further
experiments. These results showed that Ssu72 activity does not depend
on metal ions, but is rather inhibited by them. The oxidizing agent
H2O2 also impaired Ssu72 activity (Fig.
2C). Even at very low concentrations,
H2O2 abolished catalysis after a 12-min
incubation. Activity could however be recovered by adding the reducing
agent DTT, demonstrating that Ssu72 oxidation is reversible (Fig
2C).
The Signature Motif Is Required for Ssu72 Activity--
Inhibition
of Ssu72 by metal ions and by oxidation suggested a catalytic role of
the cysteine residue in the signature motif. Metal ions would mask the
cysteine side chain, with the "soft" ions Co2+,
Ni2+ and Cu2+ being more effective because of
their strong interaction with the "soft" cysteine sulfur atom or
because of an oxidizing effect in the case of Cu2+.
Consistently, H2O2 would inactivate the
cysteine side chain by oxidizing it to sulfenic acid (21). To test
directly whether residues in the signature motif are required for Ssu72
activity, we mutated cysteine 12 and arginine 18 to serine
and lysine, respectively, purified the resulting Ssu72 variants, C12S
and R18K, and subjected them to pNPP cleavage assays. The variant C12S
did not cleave pNPP even after incubation for 24 h, showing that
cysteine 12 is essential for Ssu72 activity. The variant R18K retained
only very low activity, and the catalytic reaction stopped after 10 min, so that kinetic parameters could not be determined. This points to
a crucial catalytic role of arginine 18, which does not depend solely
on the positive charge of the side chain.
Ssu72 Resembles PTPases of the Low Molecular Weight
Family--
The signature motif is found at different locations within
the polypeptide sequence of various PTPase families. In receptor-like and intracellular PTPases, the signature motif is found in the second
half of the catalytic domain, in dual specificity PTPases it is located
near the middle of the sequence, and in low Mr
PTPases it is located in the N-terminal region (22). Because the
signature motif of Ssu72 is also found near the N terminus, and because an asparagine residue within the motif is conserved between Ssu72 and
low Mr PTPases (23) but not in other PTPase
families, Ssu72 may be related to low Mr
PTPases. A relationship between Ssu72 and low
Mr PTPases is strongly supported by secondary
structure prediction (program PHD (24)), which revealed that
Ssu72 is a mixed
/
protein that shows essentially the same
succession of secondary structure elements as the low
Mr PTPases (Fig.
3; Refs. 23, 25, and 26). A comparison of
the sequences within the predicted secondary structure elements
revealed weak homology between Ssu72 and enzymes of the low
Mr family (Fig. 3), suggesting that the fold of
the proteins is the same. The signature motif is predicted to form the
loop region between the N-terminal
-strand and the subsequent
-helix, consistent with structures of low Mr
PTPases (22).

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Fig. 3.
Topological similarity of Ssu72 with low
Mr PTPases. A comparison of yeast and
human Ssu72 sequences with the sequence of bovine low
Mr PTPase is shown. Secondary structure elements
as observed in the crystal structure of bovine low
Mr PTPase are indicated above the
sequences in black (cylinders,
-helices; arrows, -strands; lines, loops).
Below the human Ssu72 sequence, the predicted secondary structure
elements (program PHD (24)) are shown in
gray. Secondary structure elements with PHD scores below 6 are shown in white. Gaps introduced in the sequence
alignments of yeast and human Ssu72 are indicated by a + sign and
arbitrary gaps due to apparent insertions or deletions in the sequences
of Ssu72 and bovine low Mr PTPase by a sign. Catalytically important residues in the signature motif
and the aspartate loop are highlighted. Note that the
alignment between Ssu72 and bovine low Mr PTPase
is tentative.
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An "Aspartate Loop" Contributes to Ssu72 Activity--
In
addition to the cysteine and arginine residues in the signature motif,
the active site of PTPases comprises a crucial aspartate residue (15,
22). In sequences of the low Mr PTPase family, this aspartate is found about 110 residues C-terminal of the signature motif (Fig. 3). In the three-dimensional structure of PTPases, the
aspartate is part of a flexible loop, here referred to as the aspartate
loop, near the entrance to the active site (23). Based on the
assumption that the fold of Ssu72 resembles that of low
Mr PTPases, we predicted a region in Ssu72 that
corresponds to the aspartate loop (Fig. 3). Indeed this region
comprises the two aspartates 140 and 143. Whereas aspartate 143 is
invariant, aspartate 140 is highly conserved and is a glutamate in
Arabidopsis thaliana Ssu72.
To test whether one of the two aspartate residues corresponds to
the active site aspartate in PTPases, we individually mutated the two
residues to alanine, purified the resulting Ssu72 variants D140A and
D143A, and quantified their activities with Michaelis-Menten kinetics.
The variant D140A showed a Km value of 7.9 mM, twice that of wild type Ssu72, and a
Vmax value 10-fold lower than that of wild type
(Table I). The variant D143A showed a 7-fold higher
Km value, but its Vmax value
was 3-fold lower than that of wild type Ssu72 (Table I). In contrast to the differences in Km values, Ssu72 wild type and
variant D143A had comparable specific activities, but the variant D140A showed a 10-fold lower specific activity than wild type (Table I).
Overall, both aspartate mutants of Ssu72 show a decreased catalytic
activity, which is reflected in the low
kcat/Km values (Table I). The
kinetic parameters suggest that aspartate 140 is important for
transition state stabilization, whereas aspartate 143 is more important
for substrate binding. These results establish the region of Ssu72
comprising aspartates 140 and 143 as the counterpart of the
catalytically important aspartate loop of PTPases.
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DISCUSSION |
Ssu72 Is a Potential Tyrosine Phosphatase--
We report here
three lines of evidence suggesting that Ssu72 is a phosphatase that
resembles PTPases. First, Ssu72 can cleave the phosphotyrosine analogue
pNPP and its catalytic activity is impaired by known PTPase-inhibiting
agents. Second, Ssu72 contains the signature motif found in all
PTPases, and conserved residues in the signature motif are required for
activity. Third, secondary structure prediction and site-directed
mutagenesis suggest that the overall structure of Ssu72 resembles that
of low Mr PTPases, including an aspartate loop
that forms part of the active site.
The Phosphatase Signature Motif Is Essential for Cell
Viability--
Published data demonstrate an essential role of the
signature motif in Ssu72 for cell viability. Truncation of an
N-terminal protein part that harbors the catalytic cysteine residue is
lethal to yeast (8). Most strikingly, a single point mutation of the catalytic cysteine residue to serine suffices to confer lethality (8).
The gene encoding for Ssu72 was originally discovered as its
ssu72-1 allele, which enhances a genetic defect in TFIIB in
yeast (27). The ssu72-1 allele gives rise to a 10-amino acid duplication in the N-terminal region of Ssu72 (8). The duplication corresponds to residues 4-15 in human Ssu72 and comprises the essential cysteine residue.
Implications for the Catalytic Mechanism--
The reaction
catalyzed by PTPases generally involves two steps and three crucial
active site residues. The CX5R signature motif
forms the phosphate-binding loop in the active site of all PTPases. The
cysteine in the CX5R motif acts as a nucleophile and accepts the PO3 moiety from the phosphotyrosine,
generating a phosphocysteine intermediate. In a second step, the
PO3 moiety is transferred to a water molecule, releasing
phosphate and regenerating the enzyme. The arginine in the
CX5R motif is required for stabilization of the
pentacovalent transition state. In addition a flexible aspartate loop
contributes an aspartate residue to the active site that serves as a
general acid/base in both steps of the reaction (13, 25, 26).
Our mutational analysis and inhibitor studies suggest that the reaction
mechanism and active site architecture of Ssu72 is similar to that of
known PTPases. Similar to PTPases (15), mutation of the cysteine
residues in the CX5R motif abolishes Ssu72
activity, and mutation of the arginine residue dramatically reduces
activity. Similar to low Mr PTPases, Ssu72 is
inhibited by vanadate, which can mimic a pentacovalent transition
state. In contrast, Ssu72 activity is not impaired by the
[BeF3]
anion, which inhibits phosphatases
that form an aspartylphosphate intermediate (19, 20). The effects of
metal ions on Ssu72 activity agree with similar studies of the PTPase
VHR (28), except that Co2+ does not impair VHR activity,
whereas it abolishes Ssu72 activity.
Differences Between Ssu72 and Low Mr PTPases--
Despite
these structural and mechanistic similarities, some substantial
differences exist between Ssu72 and low Mr
PTPases. Ssu72 differs from low Mr PTPases by
the presence of a C-terminal extension, predicted to form a
helix-loop-strand motif, and by an insertion of more than 10 residues
after the second predicted
-helix (Fig. 3). Further, a counterpart
of the aspartate loop in PTPases was identified in Ssu72, but it is
about 10 residues shorter than in low Mr PTPases
and comprises two catalytically important acidic residues instead of
one. The shorter aspartate loop in Ssu72 may be less flexible than that
of canonical PTPases, where residues in this loop can move up to 12 Å upon substrate binding (29). The structural differences in the
aspartate loops are reflected in differences in their catalytic roles.
Whereas the Ssu72 aspartate loop mutations D140A and D143A result
mainly in a modest decrease in Vmax and an
increase in Km, respectively, mutation of aspartate
129 in bovine low Mr PTPase did not change the
Km value but led to a more than 2000-fold decrease in Vmax (30). Differences in the aspartate loop
may also account for a weaker substrate affinity and a lower specific
activity of recombinant Ssu72 when compared with some low
Mr PTPases (Table I).
Possible Substrates and Functions of Ssu72--
A candidate Ssu72
substrate is the phosphorylated pol II CTD, which coordinates mRNA
transcription and processing. The CTD consists of YSPTSPS heptapeptide
repeats, which can be phosphorylated at the tyrosine 1 in mammalian
cells (31). In yeast, tyrosine 1 phosphorylation has not been reported,
but it may occur, as substitution of tyrosine 1 by phenylalanine is
lethal (32). Consistent with the idea that Ssu72 is a CTD tyrosine
phosphatase, Ssu72 mutation impairs pol II transcription but does not
affect pol I and pol III (11), both of which lack a CTD. Recombinant Ssu72, however, does not cleave synthetic CTD phosphotyrosine peptides
or isolated phosphotyrosine (not shown). Thus Ssu72 could in principle
function as a phosphotyrosine-binding protein, perhaps as an anchor
that jams CPF to phosphorylated pol II, complementing known
CTD-interacting subunits of CPF (33). Alternatively, Ssu72 phosphatase
activity may depend on the complete target protein or may be stimulated
by an activating factor, similar to the stimulation of the CTD
phosphatase Fcp1 by TFIIF (34). Ssu72 activity may also depend on
phosphorylation or on association with a small molecule, as shown for
some low Mr PTPases (15). Proteins other than
pol II could be the target of Ssu72 activity, but few
tyrosine-phosphorylated nuclear proteins are known, perhaps because of
the difficulty in detecting this transient modification. Finally, Ssu72
activity could also target nucleic acids.
Conclusion--
We have discovered an essential
phosphatase/phosphoprotein-binding activity residing in the Ssu72
protein that is intimately associated with the mRNA
transcription/processing apparatus. It is likely that this novel Ssu72
activity regulates aspects of the coupling between nuclear gene
expression machines.