From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021
Received for publication, January 22, 2001
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ABSTRACT |
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Saccharomyces cerevisiae RNA
triphosphatase Cet1 is an essential component of the yeast mRNA
capping apparatus. The active site of Cet1 resides within a
topologically closed hydrophilic RNA triphosphatase catalyzes the first step in mRNA cap
formation, the cleavage of the The binding of yeast Cet1 to Ceg1 elicits two apparently beneficial
outcomes. First, Cet1·Ceg1 interaction stabilizes the intrinsically labile guanylyltransferase activity of Ceg1 against thermal inactivation at physiological
temperatures.2
Second, the physical tethering of Cet1 to Ceg1 may facilitate recruitment of the triphosphatase to the RNA polymerase II elongation complex. Ceg1 binds in vitro and in vivo to the
phosphorylated carboxyl-terminal domain (CTD) of the largest subunit
of RNA polymerase II, whereas Cet1 by itself does not interact in
vitro with the phosphorylated CTD (3-6).
Cet1 consists of three domains: (i) a 230-aa amino-terminal segment
that is dispensable for catalysis in vitro and for Cet1 function in vivo; (ii) a protease-sensitive segment from
residues 230 to 275 that is dispensable for catalysis but essential for Cet1 function in vivo; and (iii) a catalytic domain from
residues 275 to 549 (7). A homodimeric quaternary structure for the biologically active Cet1 protein was inferred from analysis of the
purified recombinant enzyme by glycerol gradient sedimentation and then
confirmed by x-ray crystallography (7, 8). A Cet1·Ceg1 capping enzyme
complex reconstituted in vitro from separately purified
components is surmised from velocity sedimentation analysis to be a
heterotrimer consisting of two molecules of triphosphatase and one
molecule of guanylyltransferase (7).
Is homodimer formation essential for Cet1 function? Deletion analysis
shows that the carboxyl-terminal domain Cet1(276-549) has a monomeric
quaternary structure, yet it retains full catalytic activity in
vitro (7). Thus, homodimerization is not essential for catalysis.
However, the monomeric domain by itself cannot support yeast cell
growth, even when it is overexpressed at high gene dosage under the
control of a strong promoter. Interpretation of the deletion data is
complicated by the fact that an amino-terminal truncation to position
275 also removes the guanylyltransferase-binding site
243WAQKW247, which is located on the
protein surface (9) and is responsible for Cet1-mediated stabilization
of the guanylyltransferase Ceg1.2
Remarkably, the in vivo function of Cet1(276-549) is
completely restored when the monomeric triphosphatase domain is fused in cis to the guanylyltransferase domain of the mouse
capping enzyme (7). The mouse domain, Mce1(211-597), binds avidly to the phosphorylated CTD (10) and can thereby act as a vehicle to deliver
the fused RNA triphosphatase to the RNA polymerase II elongation
complex (7, 11). Also, because the mouse guanylyltransferase is
thermostable (unlike Ceg1), the chimeric capping enzyme bypasses the
need for the Ceg1-stabilization function of the
243WAQKW247 peptide of Cet1.2
To focus specifically on the role of homodimerization in Cet1 function
in vivo, we have initiated a mutational analysis of the
amino acids comprising the homodimer interface revealed by the crystal
structure (8). Alanine-cluster mutations were introduced into the
biologically active protein Cet1(201-549), which contains both the
guanylyltransferase-binding and catalytic domains (7). The results of
this analysis indicate that homodimerization of yeast RNA
triphosphatase is important for its function in vivo.
Mutagenesis of Yeast RNA Triphosphatase--
Alanine-cluster
mutations were introduced into the CET1(201-549) gene by
polymerase chain reaction (12). The mutated genes were inserted into
the yeast CEN TRP1 plasmid pCET1-5'3', where expression of
the inserted gene is under the control of the natural CET1
promoter (13). The inserts were sequenced completely to exclude the
acquisition of unwanted mutations during amplification and cloning. The
genes were excised from their respective pCET1-5'3' plasmids with
NdeI and BamHI and inserted into the yeast
expression vector pYN132 (CEN TRP1). In this vector,
expression of CET1(201-549) is driven by the strong
constitutive yeast TPI1 promoter.
NdeI/BamHI restriction fragments containing the
mutated genes were also cloned into the multicopy expression plasmid
pYX232 (2µ TRP1) with triphosphatase expression being
driven by the TPI1 promoter. The in vivo activity of the mutated CET1 alleles was tested by plasmid shuffle.
Yeast strain YBS20 (trp1 ura3 leu2
cet1::LEU2 p360-Cet1[CEN URA3
CET1]) was transformed with TRP1 plasmids containing
the wild-type and mutant alleles of CET1(201-549). Trp+
isolates were selected and then streaked on agar plates containing 0.75 mg/ml 5-fluoroorotic acid (5-FOA). Growth was scored after 7 days of
incubation at 25, 30, and 37 °C or 14 days at 14 °C. Lethal
mutants were those that failed to form colonies on 5-FOA at any
temperature. Individual colonies of the viable CET1 mutants
were picked from the FOA plate at permissive temperature and patched to
YPD agar. Two isolates of each mutant were tested for growth on YPD
agar at 14, 25, 30, and 37 °C. Growth was assessed as follows: +++
indicates colony size indistinguishable from strains bearing wild-type
CET1(201-549); ++ denotes slightly reduced colony size; + indicates that only pinpoint colonies were formed.
Purification and Activity of Recombinant Yeast
Triphosphatase--
NdeI/BamHI fragments
encoding mutated versions of Cet1(201-549) were excised from the
respective pCET1-5'3' plasmids and inserted into pET16b. Wild-type
Cet1(201-549) and the F272A-L273A and D279A-D280A mutants were
expressed in Escherichia coli BL21(DE3) at 18 °C by
isopropyl-1-thio-
Triphosphatase reaction mixtures (10 µl) containing 50 mM
Tris HCl (pH 7.5), 5 mM DTT, 2 mM
MnCl2, 1 mM [ Glycerol Gradient Sedimentation--
Aliquots (30 µg) of the
nickel-agarose preparations of the wild-type and mutant Cet1(201-549)
proteins were mixed with BSA (25 µg), and cytochrome c (25 µg) in 0.2 ml of buffer G (50 mM Tris HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 2 mM DTT,
0.05% Triton X-100). The mixtures were layered onto 4.8-ml 15-30%
glycerol gradients containing buffer G. The gradients were centrifuged in a Beckman SW50 rotor at 50,000 rpm for 25 h at 4 °C.
Fractions (~0.2 ml) were collected from the bottoms of the tubes.
Aliquots (20 µl) of odd-numbered fractions were analyzed by SDS-PAGE
along with samples of the input protein mixtures for each gradient. Polypeptides were visualized by staining with Coomassie Blue dye. Aliquots (1 µl) of each fraction were assayed for triphosphatase activity as described above.
Structure-based Mutational Analysis of the Cet1 Homodimer
Interface--
Yeast RNA triphosphatase crystallized as a homodimer
(Fig. 1A). Each protomer is
composed of four
The Cet1 dimer interface (colored green in Fig. 1) is
extensive, with a buried surface area of 1860 Å2 per
protomer. Elements that comprise the dimer interface are strands
To gauge the role of homodimerization in Cet1 function, we performed
alanine-cluster mutagenesis of pairs of vicinal amino acids. The
double-Ala mutations were introduced into the biologically active
Cet1(201-549) protein. A total of 42 residues (17% of the polypeptide) were changed to alanine in this analysis, 24 of which were
constituents of the crystallographic dimer interface. The cluster
mutations also embraced residues in helices Mutational Effects in Vivo--
The
CET1(201-549)-Ala/Ala genes were cloned into a CEN
TRP1 vector under the control of the natural CET1
promoter and then tested by plasmid shuffle for their ability to
complement a cet1
Four other Ala-cluster mutants displayed a conditional phenotype,
whereby they gave rise to 5-FOA-resistant colonies at low temperatures
but failed to yield colonies at high temperatures. These
CET1(201-549)-Ala/Ala strains were isolated from 5-FOA
plates at permissive temperature and then tested for growth on rich
medium (YPD) at 14, 25, 30, and 37 °C. The D287A-W288A
strain grew as well as wild-type yeast at 14 °C (scored as +++), but
formed small colonies at 25 °C (scored as ++), pinpoint colonies at
30 °C (scored as +), and failed to grow at 37 °C (
The remaining 13 double-Ala mutants yielded 5-FOA-resistant colonies at
all temperatures and grew as well as the isogenic CET1(201-549) "wild-type" strain on YPD agar at 14, 25, 30, and 37 °C. We infer that the 26 side chains that were
substituted in the fully viable mutants are unimportant per
se for Cet1 function in vivo. These nonessential
residues (highlighted by plus signs over the Cet1 sequence
in Fig. 2) included 15 amino acids that comprise the crystallographic
dimer interface (Ile268, Asp269,
Pro270, Pro277, Ser284,
Pro325, Val326, Ser327,
Ser328, Phe332, Thr333,
Ile358, Lys367, Phe368, and
Asn526). The Ala-cluster mutants that were fully viable
were not analyzed further.
Effects of Increased Gene Expression and Gene Dosage on the Mutant
Phenotypes--
The 16 residues substituted in the 8 Ala-cluster
alleles that were lethal or ts when expressed in single copy
from the natural CET1 promoter are highlighted by
dots above the Cet1 sequence in Fig. 2. Six of the eight
Ala-clusters entailed substitution of at least one component of the
crystallographic dimer interface. If homodimerization is important
in vivo, we reasoned that the growth phenotypes elicited by
mutations that diminish the dimerization equilibrium constant might be
ameliorated by increasing the expression of the mutant protein, in
effect attaining a threshold level of dimeric Cet1 via mass action.
Also, if some of the mutations reduced triphosphatase activity to a
level below the threshold required for growth, then increased gene
expression might restore activity to a supra-threshold level. To
explore these scenarios, the lethal and ts Ala-cluster
alleles were retested for biological activity in single-copy when their
expression was driven by the strong constitutive yeast TPI1
promoter instead of the CET1 promoter.
Increased promoter strength completely suppressed the ts
phenotype of D287A W288A such that the mutant strain
grew as well as the CET1 strain on YPD agar at 37 °C
(Table II). Changing promoters partially
suppressed the ts phenotypes of F272A-L273A,
I470A-I472A, and I529A-I530A. Partial suppression
was manifest as an upward shift in the restrictive growth temperatures.
For example, I470A-I472A under TPI1 control
displayed +++ growth at 30 °C, compared with no growth under its
natural promoter. F272A-L273A grew at 30 and 37 °C
(albeit slowly) and I529A-I530A was able to grow at 30 °C (Table II). Increased promoter strength did not reverse the lethality of the D279A-D280A, C330A-V331A, or
F523A-L524A mutations. However, it did restore partial
function to the L519A-I520A allele, which was capable of
supporting growth at 14 °C, albeit not at 25 °C or higher
(Table II).
In light of these effects, we proceeded to transfer the
TPI-CET1-Ala/Ala alleles to high-copy 2µ vectors and to
test the effects of increased gene dosage on their in vivo
activities. This maneuver abated the lethality of
F523A-L524A, which displayed +++ growth at 14 °C, but no
growth at 25 °C or higher; it also improved the growth of
L519A-I520A at 14° and 25 °C (Table II). The other
mutational effects were unaffected by increased gene dosage; in
particular, the D279A-D280 and C330A-V331A
alleles were lethal even in high copy.
Rescue of Yeast RNA Triphosphatase Mutations by Fusion to Mouse
Guanylyltransferase--
Even a monomeric version of yeast RNA
triphosphatase is functional in vivo when it is fused to the
guanylyltransferase domain of the mouse capping enzyme, provided that
it retains phosphohydrolase catalytic activity (7). Thus, a functional
test of chimeric capping enzymes containing Cet1(201-549)-Ala/Ala
mutants fused to the mouse guanylyltransferase can discriminate
genetically whether the complete or partial loss of function elicited
by the Ala-cluster mutations is caused by a catalytic defect (be it a global folding problem or a direct perturbation of the active site) or
a defect in ancillary functions uniquely required in yeast cells
containing only the endogenous guanylyltransferase Ceg1. In the latter
scenario, we would expect the fusion maneuver to restore function
to the Cet1(201-549)-Ala/Ala mutant, whereas no salutary effects are
expected in the former case.
CET1(201-549)-Ala/Ala-MCE1(211--
597) chimeras were cloned
into CEN TRP1 vectors under the control of the
TPI1 promoter and then tested by plasmid shuffle for
complementation of cet1
In addition, we found that the other Ala-cluster mutants that displayed
ts growth defects when expressed by themselves in single
copy under the control of the TPI1 promoter were suppressed completely or partially by fusion to the mouse guanylyltransferase. Of special note was F272A-L273A, which was fully functional
at all temperatures as a chimeric enzyme. The Phe272 and
Leu272 side chains are components of the hydrophobic dimer
interface (Fig. 1C), and we surmise from the genetic
evidence that the phenotype of this mutant is likely caused by a
dimerization defect. On the other hand, the I529A-I530A
chimera supported growth at 30 °C, but not at 37 °C, whereas
growth of the L519A-I520A and F523A-L524A chimeras was still severely ts. Apparently, these
Ala-cluster mutants are defective in aspects other than, or in addition
to, Cet1 homodimerization.
Biochemical Characterization of Mutant Enzymes--
We produced
the mutated Cet1(201-549) proteins F272A-L273A and D279A-D280A in
bacteria as His10-tagged fusions and purified them from
soluble bacterial lysates by nickel-agarose chromatography. Wild-type
His10-Cet1(201-549) was purified in parallel. SDS-PAGE analysis showed that the ~44-kDa Cet1(201-549) protein was the predominant species in each enzyme preparation (Fig.
4A). Phosphohydrolase activity
was assayed by the release of 32Pi from
[
The native sizes of the recombinant triphosphatases were investigated
by sedimentation through 15-30% glycerol gradients. Marker proteins
BSA and cytochrome c were included as internal standards.
After centrifugation, the polypeptide compositions of the odd-numbered
gradient fractions were analyzed by SDS-PAGE. The sedimentation profile
for wild-type Cet1(201-549) is shown in Fig.
5. The triphosphatase (44 kDa) sedimented
as a discrete peak coincident with BSA (68 kDa), consistent with the
wild-type enzyme being an asymmetric homodimer (8). The triphosphatase activity profile paralleled the distribution of the Cet1 polypeptide (not shown).
The sedimentation profiles for the F272A-L273A and D279A-D280A proteins
are shown in Fig. 5. Both mutant proteins sedimented at positions
between BSA and cytochrome c, suggesting that
F272A-L273A and D279A-D280A are monomers. The triphosphatase activity
profiles of both mutants paralleled the distributions of the Cet1
polypeptide in the gradient (not shown). Note that the present
biochemical evidence that the peptide segment containing
Phe272 and/or Leu273 is important for
homodimerization of Cet1 consolidates our earlier finding that
amino-terminal deletion of Cet1 up to residue 275 results in a
catalytically active, monomeric enzyme (7).
The present study establishes the importance of homodimerization
for the in vivo function of yeast RNA triphosphatase Cet1 and it highlights the contributions of two separate facets of the dimer
interface, both of which play a role in Cet1 function in
vivo. We also identify key residues in the hydrophobic core of the
Cet1 protomer that stabilize the triphosphatase in vivo.
Structural Interpretations of the Mutational Effects--
One of
the key facets of the interface entails hydrophobic interactions of the
side chains of strand
The second functionally relevant dimer interface involves hydrophobic
side-chain interactions between
The receiving hydrophobic pocket on the dimer partner for the pre-
The side chains of Ile529 and Ile530 make
intimate cross-dimer contacts with Phe272 and
Leu273. Note that the conditional in vivo
phenotypes of I529A-I530A in the various promoter and fusion
contexts tested were roughly similar to those of
F272A-L273A, except that I529A-I530A function at
37 °C was not restored by fusion to mouse guanylyltransferase. Although Ile529 does not contact any other residues besides
Phe272, Leu273, and Pro277 in the
dimer partner, Ile530 makes additional intramolecular
contributions to the hydrophobic core of the Cet1 protomer (involving
Val493 and Leu495 in
The I470A-I472A (
We propose that the L519A-I520A (
Similarly, the lethal F523A-L524A mutation, which is
minimally suppressed even in high copy, is almost certainly caused by effects on active site architecture rather than quaternary structure, because Phe523 and Leu534 are oriented toward
the hydrophobic core of the Cet1 protomer and they make no
contributions to the crystallographic dimer interface. Rather,
Phe523 is situated in a hydrophobic-aromatic-hydrophobic
sandwich between Met308 (in
Finally, the conditional phenotype of D287A-W288A ( Why Is Dimerization Important?--
Although homodimerization in
clearly important for Cet1 function in vivo, the rationale
for assembly of a dimeric triphosphatase with two parallel tunnels is
still not clear. We suspect that mRNA modification in
vivo does not require two functional triphosphatase active sites
in the same complex, insofar as overexpression in yeast of a
catalytically inactive triphosphatase mutant that still homodimerizes
and binds to the guanylyltransferase Ceg1 does not elicit a dominant
negative phenotype.4 The
mutations characterized here that specifically affect homodimerization would not alter the high affinity guanylyltransferase-binding site on
the surface of Cet1. The peptide motif Cet1(232-265) is sufficient
per se to bind Ceg1 in solution and to stabilize the guanylyltransferase against thermal inactivation.
There are two potential Ceg1-binding sites on the triphosphatase dimer
located on opposite sides of the structure. Sedimentation analysis
suggests that the dimeric triphosphatase binds 1 molecule of Ceg1
(which is itself a monomer in solution) to form a heterotrimeric capping enzyme complex (7). The available data do not exclude the
possibility that the reconstituted complex is an asymmetrically shaped
(Cet1)2-(Ceg1)2 tetramer, and it is conceivable
that the dimeric triphosphatase could bind two Ceg1 monomers if Ceg1
were present in excess during the reconstitution. In a strictly
heterotrimeric model, it is possible that binding of Ceg1 to one
surface of the Cet1·Cet1 dimer sterically precludes binding of a
second Ceg1p on the opposite face (7). It is also conceivable that Ceg1 bound to the 232-265 peptide in one protomer of Cet1 makes cross-dimer interactions with the dimer partner that are relevant to cap formation. Perhaps the interaction of the Cet1 dimer with Ceg1 creates a binding
site for nascent RNA that permits transit of the 5' terminus from the
triphosphatase active site to the guanylyltransferase active site
without complete release of the polynucleotide between the catalytic
steps. We anticipate that a crystal structure of yeast triphosphatase
bound to yeast guanylyltransferase will provide a definitive account
of the stoichiometry of the Cet1·Ceg1 complex and shed some light on
the rationale for the distinctive homodimeric structure of the
triphosphatase component.
-barrel (the triphosphate tunnel)
that is supported by a globular hydrophobic core. The homodimeric
quaternary structure of Cet1 is formed by a network of contacts between
the partner protomers. By studying the effects of alanine-cluster
mutations, we highlight the contributions of two separate facets of the
crystallographic dimer interface to Cet1 function in vivo.
One essential facet of the interface entails hydrophobic cross-dimer
interactions of Cys330 and Val331 and a
cross-dimer hydrogen bond of Asp280 with the backbone amide
of Gln329. The second functionally relevant dimer
interface involves hydrophobic side-chain interactions of
Phe272 and Leu273. Ala-cluster mutations
involving these residues elicited lethal or severe
temperature-sensitive phenotypes that were suppressed completely by
fusion of the mutated triphosphatases to the guanylyltransferase domain
of mammalian capping enzyme. The recombinant D279A-D280A and
F272A-L273A proteins retained phosphohydrolase activity but sedimented
as monomers. These results indicate that a disruption of the dimer
interface is uniquely deleterious when the yeast RNA triphosphatase
must function in concert with the endogenous yeast guanylyltransferase.
We also identify key residue pairs in the hydrophobic core of the Cet1
protomer that support the active site tunnel and stabilize the
triphosphatase in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
phosphoanhydride bond of
5'-triphosphate RNA to yield a diphosphate end that is then capped with
GMP by RNA guanylyltransferase (1). The budding yeast
Saccharomyces cerevisiae encodes separate triphosphatase
(Cet1; 549 aa1) and
guanylyltransferase (Ceg1; 459 aa) proteins that interact in
trans to form a heteromeric capping enzyme complex. Yeast
cell growth depends on the catalytic activities of both enzymes and their physical interaction.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside induction for
20 h in the presence of 2% ethanol (2). The proteins were
purified from soluble bacterial lysates by nickel-agarose
chromatography as described previously (2, 7). The 0.2 M
imidazole eluate fractions containing Cet1(201-549) were dialyzed
against 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM DTT, 10% glycerol, 0.05% Triton X-100, then stored at
80 °C.
-32P]ATP, and
Cet1(201-549) as specified were incubated for 15 min at 30 °C. The
reactions were quenched by adding 2.5 µl of 5 M formic
acid. An aliquot of the mixture was applied to a
polyethyleneimine-cellulose TLC plate, which was developed with 0.5 M LiCl and 1 M formic acid. The release of
32Pi from [
-32P]ATP was
quantitated by scanning the TLC plate with a PhosphorImager.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helices and 11
strands. The secondary
structure elements are displayed over the Cet1 protein sequence in Fig.
2. The striking feature of the tertiary
structure is the formation of a topologically closed tunnel composed of 8 antiparallel
strands. In the dimer, the two tunnels are parallel and oriented in the same direction. A surface view of the monomer is
shown in Fig. 1A looking into the tunnel. Rotation of the
molecule provides a side view that highlights a platform-like structure in front of the tunnel entrance (Fig. 1B). The
triphosphatase active site resides within the tunnel. The
guanylyltransferase-binding site is located on the surface and is
colored red in Fig. 1.
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Fig. 1.
The homodimer interface of yeast RNA
triphosphatase. A, the triphosphatase dimer is
shown with one protomer as a space-filling surface image
(top) and the dimer partner as a worm trace of the
polypeptide (bottom). The view is looking into the tunnel
entrance. The red surface represents the domain responsible
for binding of Cet1 to the yeast guanylyltransferase Ceg1. The
Cet1·Cet1 dimer interface is depicted in green.
B, side view of the dimer after rotation ~90° to the
right. C, view of the structure oriented to highlight the
dimer interface. The Phe272 side chain is shown on the worm
representation projecting into a deep surface pocket on the dimer
partner.
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Fig. 2.
Structure-based mutational analysis. The
secondary structure of S. cerevisiae Cet1 from residues 241 to 539 is shown above its amino acid sequence, which is aligned to the
homologous segment of the Candida albicans RNA
triphosphatase CaCet1. Gaps in the alignment are indicated by
dashes. Amino acid pairs in Cet1 that were mutated to
alanine in the present study and found to have no effect on
CET1 function in vivo are denoted by plus
signs above the sequence. The positions at which Ala-cluster
mutations elicited lethal or ts phenotypes are denoted by
dots.
2
and
3, helices
1 and
4, the loop immediately preceding
1,
the loop between
9 and
10, and the loop between
3 and
4. The molecular contacts of the dimer interface are listed in Fig. 3. These entail multiple hydrophobic
interactions and a network of side-chain and main-chain hydrogen bonds.
The hydrophobic core of the dimer interface is stabilized by
interactions of residues Ile268, Phe272,
Leu273, and Pro277 (located immediately
proximal to
1) with hydrophobic residues on the dimer partner
located in the segments from 354-368, 463-467, and 519-530 (Fig. 3).
Indeed, the aromatic side chain of Phe272 inserts into a
small hydrophobic pocket on the surface of the dimer partner (Fig.
1C). Additional hydrophobic interactions occur between side
chains of strand
2 of one protomer (325) and strand
3 in the
partner (330) (Table I). Main-chain
hydrogen bonds between
2 and
3 form an antiparallel sheet at the
dimer interface.
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Fig. 3.
Functional dimer interactions defined by
mutagenesis. A summary of the two main dimer interactions in the
Cet1 crystal is shown (8). Side chains at which alanine substitutions
elicited no growth phenotype are in italics. Side chain
interactions embraced by lethal or conditional Ala-cluster mutations
are highlighted in shaded boxes. Positions for which
mutational data are not available and all of the main-chain contacts
are listed in plain text. All of the alanine-scanning
results are from the present study, except for the H463A
mutation (11).
Effects of alanine-cluster mutations on Cet1 function in vivo
, colonies failed to grow.
1 and
4 that
comprise the hydrophobic core of the Cet1 protomer upon which the
triphosphate tunnel rests.
strain of S. cerevisiae. The
results are summarized in Table I. Growth of cet1
is contingent upon maintenance of a wild-type CET1 allele on
a CEN URA3 plasmid. Therefore, the cet1
strain is unable to grow on agar medium containing 5-FOA (5-fluoroorotic acid,
a drug which selects against the URA3 plasmid) unless it is
first transformed with a biologically active RNA triphosphatase gene on
the TRP1 plasmid. Trp+ CET1(201-549)-Ala/Ala
transformants were tested for growth on 5-FOA. Triphosphatase mutations
were judged to be lethal if they failed to support colony formation on
5-FOA after prolonged incubation at four different temperatures (14, 25, 30, and 37 °C). Four of the Ala-cluster alleles were lethal by
this criterion: D279A-D280A, C330A-V331A,
L519A-I520A, and F523A-L524A.
growth). The
F272A-L273A mutant grew slowly at 14 °C and not at all at
30 °C or higher. The I470A-I472A and
I529A-I530A mutations elicited the most severe conditional
phenotypes, with weak growth at 14 °C and no growth at 25 °C or
higher (Table I).
Suppression of Cet1 mutational effects by overexpression or fusion to
mouse guanylyltransferase
, colonies failed to grow.
. The salient findings were that
the D279A-D280A and C330A-V331A alleles, which
were lethal even when expressed at high gene dosage, were fully
functional at all temperatures when fused to the mouse
guanylyltransferase (Table II). We surmise that these two Ala-cluster
mutations do not significantly affect phosphohydrolase catalytic
activity in vivo. This conclusion is consistent with the
distant location of the four mutated residues from the enzyme's active
site in the crystal structure (8). Because the Asp280,
Cys330, and Val331 side chains are components
of the homodimer interface, we infer that the lethal phenotypes of the
cluster mutations reflect a requirement for Cet1 homodimerization for
RNA processing by the yeast capping apparatus.
-32P]ATP in the presence of manganese chloride (2).
The extents of ATP hydrolysis increased as a function of input enzyme
for each protein (Fig. 4B). A specific activity for the
wild-type Cet1(201-549) of 0.58 nmol of ATP hydrolyzed per nanogram of
protein in 15 min was calculated from the slope of the titration curve in the linear range. (This value translates into a turnover number of
29 s
1). The specific activity of F272A-L273A was
virtually identical to that of the wild-type enzyme, whereas
D279A-D280A was 30% as active as wild-type. The remaining lethal or
ts Ala-cluster mutants were insoluble when produced in
bacteria and thus not amenable to biochemical characterization.
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Fig. 4.
Purification and triphosphatase activity of
Cet1 Ala-cluster mutants. A, aliquots (4 µg) of the
nickel-agarose preparations of wild-type (WT) Cet1(201-549)
and the Ala-cluster mutants F272A-L273A (FL) and D279A-D280A
(DD) were analyzed by SDS-PAGE. Polypeptides were visualized
by staining with Coomassie Blue dye. The positions and sizes (in kDa)
of marker proteins are indicated on the left. B,
triphosphatase activity was assayed as described under "Experimental
Procedures". 32Pi release is plotted as a
function of input protein for each enzyme preparation. Each datum is
the average of two assays.
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Fig. 5.
Glycerol gradient sedimentation.
Sedimentation was performed as described under "Experimental
Procedures." The glycerol gradient fractions were analyzed by
SDS-PAGE. Coomassie Blue-stained gels are shown. Gradient fraction
numbers are specified above the lanes. The input protein
mixtures are analyzed in lane L. The identities of the
polypeptides are indicated on the left.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 of one protomer and strand
3 in the other
protomer, of which Cys330 and Val331 appear to
be the most critical constituents. This interface also embraces
Asp280 (in
1), which engages in a cross-dimer hydrogen
bond with the backbone amide of Gln329 (in the turn
connecting
2 and
3). Two Ala-cluster mutations involving these
residues (D279A-D280A and C330A-V331A) elicited lethal phenotypes that
could not be suppressed by overexpression of the mutated
triphosphatases but were suppressed completely by fusion of the mutated
enzymes to the mammalian RNA guanylyltransferase. These results
indicated that a disruption of the dimer interface is uniquely
deleterious when the yeast RNA triphosphatase must function in
concert with the yeast guanylyltransferase Ceg1. Biochemical analysis
of the recombinant D279A-D280A protein confirms that its
phosphohydrolase activity was intact and that the mutant protein sedimented as a monomer. Thus, we attribute the in vivo
phenotypes of D279A-D280A to an isolated defect in
homodimerization. Although biochemical analysis of C330A-V331A was
hampered by insolubility of the recombinant mutant protein, the
in vivo phenotypes are consistent with a dimerization
defect. Mutations of several other side chains (Pro325,
Val326, Ser327, Ser328,
Phe332, and Thr333) that make up this segment
of the crystallographic dimer interface did not elicit effects on cell
growth, implying that their individual contributions are subtle at
best. Of course, some these residues also engage in main-chain contacts
to the dimer partner, but the significance of these interactions may
not be revealed by replacing the side chain with alanine.
4 and residues Phe272
and Leu273 in the loop preceding
1. Replacement of the
Phe272 and Leu273 side chains results in a
catalytically active monomeric enzyme that confers a severe
ts growth phenotype when expressed under its native
promoter. The growth defect is ameliorated in part by increasing the
expression level and suppressed completely by fusion to mouse
guanylyltransferase. Alanine mutations of neighboring dimer interface
residues Ile268, Asp269, Pro270,
and Pro277 did not affect cell growth. However, as noted
above, we cannot judge from these results whether or not the main chain
contacts of residues 269 and 270 contribute to Cet1 function in
vivo. A related issue is whether the growth defect of the
F272A-L273A mutant might result from an indirect effect of
alanine substitution on the cross-dimer hydrogen bond of the main chain
amide of residue 272 (Fig. 3). If this were the case, then we would
expect the loss of the hydrogen-bonding partner (the side chain O
of
Asn526) to phenocopy F272A-L273A. Because the
replacement of Asn526 by alanine elicited no phenotype, we
surmise that the F272A-L273A phenotypes reflect the
contributions of the hydrophobic side chains to the dimer interface
rather than those of the main chain.
1
loop is composed primarily of residues in
4. Although the
N525A-N526A cluster mutation of
4 did not affect cell
growth, the three other cluster mutants in
4
(L519A-I520A, F523A-L524A, and
I529A-I530A) had either lethal or severely conditional
phenotypes. To what extent are these mutant phenotypes attributable to
defects in homodimerization?
11) that supports the
floor of the triphosphate tunnel. The neighboring residues
Glu492 and Glu494 of
11 bind the metal
cofactor in the active site of Cet1. Thus, we suspect that the I530A
mutation imposes conformational effects on the structure of the tunnel
in addition to its effects on homodimerization and that the
homodimerization problem is overcome in the yeast-mouse chimera whereas
the conformational effects on the tunnel are not. This model is
consistent with our isolation in a genetic screen of a
temperature-sensitive yeast mutant (cet1-1) that contains a
single mutation of Ile530 to threonine (7). Furthermore,
directed mutagenesis of
11 showed that the single alanine mutations
of Val493 or Leu495 (the residues contacted
intramolecularly by Ile530) resulted per se in a
ts growth defect at 37 °C (11).
10) mutant phenotype is also likely to
reflect more than one underlying process, including (i)
diminished dimerization affinity caused by the alanine substitution for
Ile470 (which interacts with Phe272 on the
dimer partner) and (ii) intramolecular effects of the I472A mutation on
the conformation of
10, which comprises part of the floor and
lateral wall of the triphosphate tunnel. Ile472 makes van
der Waals contacts with Phe348 and Ile355 in
2, and this helix in turn stabilizes the
strands of the tunnel
wall.
10 contains three hydrophilic side chains (Arg269,
Asp471, and Thr473) immediately surrounding
Ile470 and Ile472, which are essential for the
phosphohydrolase activity of
Cet1.3
4) phenotype,
i.e. lethality when expressed by its own promoter and a
severe ts growth defect when overexpressed and in the
context of the mouse guanylyltransferase fusion, is caused
predominantly by intramolecular effects of one or both alanine changes
on the stability of the triphosphate tunnel. Leu519 makes
van der Waals contacts with Ile497 in
11 in the same
protomer. The cross-dimer interaction of Leu519 with
Ile268 is probably not relevant to the ts
phenotype insofar as alanine substitution for Ile268 was
itself without effect. Note that the temperature-sensitive cet1-15 strain isolated previously in a genetic screen
contains a single mutation of Leu519 to proline (7).
1) and Ile497
(in
11) that likely imparts stability to the floor of the tunnel.
1),
which was suppressed completely by increasing promoter strength alone, is probably unrelated to effects on dimerization, insofar as neither Asp287 nor Trp288 contributes to the
crystallographic dimer interface. However, Trp288 is a key
component of the hydrophobic core of the Cet1 protomer. The aromatic
Trp288 side-chain stacks on Leu415 (in
7)
and makes van der Waals contacts with Ala413 (
7) and
Ile428 (
8) that likely stabilize the floor and lateral
wall of the triphosphate tunnel. The temperature-sensitive
cet1-14 strain isolated previously in a genetic screen
contains a single mutation of Trp288 to arginine (7).
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ACKNOWLEDGEMENT |
---|
We are grateful to Chris Lima for helpful discussions and comments on the manuscript.
![]() |
FOOTNOTES |
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* This work was supported by NIH 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: Molecular
Biology Program, Sloan-Kettering Institute, 1275 York Ave., New York, NY 10021. Tel.: 212-639-7145; Fax: 212-717-3623; E-mail:
s-shuman@ski.mskcc.org.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M100588200
2 S. Hausmann, C. K. Ho, and S. Shuman, manuscript in preparation.
3 M. Bisaillon and S. Shuman, unpublished.
4 Y. Pei and S. Shuman, unpublished.
![]() |
ABBREVIATIONS |
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The abbreviations used are: aa, amino acids; CTD, carboxyl-terminal domain; 5-FOA, 5-fluoroorotic acid; DTT, dithiothreitol; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis.
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