From the
The yeast S-II null mutant is viable, but the mutation induces
sensitivity to 6-azauracil. To examine whether the region needed for
stimulation of RNA polymerase II and that for suppression of
6-azauracil sensitivity in the S-II molecule could be separated, we
constructed various deletion mutants of S-II and expressed them in the
null mutant using the GAL1 promoter to see if the mutant
proteins suppressed 6-azauracil sensitivity. We also expressed these
constructs in Escherichia coli, purified the mutant proteins
to homogeneity, and examined if they stimulated RNA polymerase II. We
found that a mutant protein lacking the first 147 amino acid residues
suppressed 6-azauracil sensitivity but that removal of 2 additional
residues completely abolished the suppression. A mutant protein lacking
the first 141 residues had activity to stimulate RNA polymerase II,
whereas removal of 10 additional residues completely abolished this
activity. We also examined arrest-relief activity of these mutant
proteins and found that there is a good correlation between RNA
polymerase II-stimulating activity and arrest-relief activity.
Therefore, at least the last 168 residues of S-II are sufficient for
expressing these three activities.
Transcription factor S-II, originally isolated from Ehrlich
ascites tumor cells as a specific stimulator of RNA polymerase II, has
been studied extensively
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) .
Results have shown that S-II is one of the transcription elongation
factors that promote read-through by RNA polymerase II of pausing sites
within genes in vitro (16, 17) . S-II has been
proposed to release pausing by inducing cleavage of the 3`-end of the
nascent transcript in the ternary elongation complex (Refs.
18-24; for review, see Ref. 25).
Recently, we purified S-II
from yeast ( Saccharomyces cerevisiae) and determined its
complete amino acid sequence by isolating its gene
(26) .
Significant sequence similarity was found between yeast S-II and other
S-IIs so far sequenced
(10, 27, 28) . Yeast S-II
specifically stimulated yeast RNA polymerase II
(26) and was
shown to promote cleavage and elongation of nascent RNA in the
elongation complex of transcription in vitro (29, 30) . Therefore, like the S-IIs of higher
eukaryotes, yeast S-II is a transcription elongation factor.
The
nucleotide sequence of the yeast S-II gene indicated that it is the
same protein as STP
Plasmids containing S-II deletion mutants were
constructed as follows. S-II deletion mutants were amplified by
polymerase chain reaction using the S-II gene (pYSII-2) as a template,
and the resulting DNA fragments were ligated to the GAL1 promoter in pYO324. Mutant S-II proteins were induced by
galactose.
Recombinant
S-II mutant proteins were purified as follows. Freshly harvested E.
coli cells were lysed by treatment with lysozyme-EDTA and
deoxycholate, and the lysate was centrifuged at 100,000
The S-II null
mutants that we established previously required uracil
(26) , so
they could not be used for testing suppression of 6-azauracil
sensitivity. Therefore, we constructed a new S-II null mutant. Southern
blot analysis of genomic DNA derived from TNY14 showed that its S-II
gene was disrupted. We further confirmed that S-II is not present in
TNY14 by an immunofluorescence study with affinity-purified anti-S-II
antibody. Immunofluorescence was exclusively detected in the nuclei of
wild type yeast, whereas no immunofluorescence was detected in TNY14
(data not shown). Thus, we concluded that S-II is a nuclear protein and
that TNY14 lacks this protein.
TNY14 was sensitive to 6-azauracil
and did not form colonies on MVD agar plates containing 100 µg/ml
of 6-azauracil (Fig. 1, A and B). When pYSG6 (an
expression vector containing the GAL1 promoter-yeast S-II gene
coding region) was introduced into TNY14, the resulting transformant
could form colonies on MVGS agar plates in the presence of 6-azauracil
(Fig. 1, C and D), indicating that introduction
of full-length S-II cDNA suppressed the 6-azauracil sensitivity of
TNY14. However, it did not form colonies on MVD plates as expected.
Using this system, we examined the suppressions of 6-azauracil
sensitivity by various S-II deletion mutants.
On the bases of these
results, we constructed seven clones of S-II deletion mutants. Of
these, five were deletion mutants of the amino-terminal side, and two
were those of the carboxyl-terminal side. We found that the
amino-terminal mutants
Using various S-II deletion mutants of yeast, we demonstrated
that at least 1-147 residues from the amino-terminal are not
essential, and the last 162 residues are sufficient for suppression of
the 6-azauracil sensitivity of an S-II null mutant. We also showed that
for activity to stimulate RNA polymerase II and relieve arrest, there
is a critical point in S-II between residues 142 and 151, and that 168
but not 158 of the carboxyl-terminal residues were necessary for
stimulation of RNA polymerase II and relief of transcription arrest
in vitro. Therefore, this region should contain binding
regions for both RNA polymerase II and nucleic acid
(6, 42, 43) . For technical reasons, we could
not purify the
Unlike mutants lacking regions of the amino-terminal
half, those lacking about 50 residues from the carboxyl-terminal were
shown to have lost all the stimulatory, arrest-relief, and suppressive
activities. Again, we could not examine these activities with a single
mutant for technical reasons, but we deduced that the
These genetical and biochemical results
strongly suggest that 6-azauracil sensitivity is due to loss of
function of S-II as a transcription factor. There seem to be two
possible reasons why the S-II null mutant has acquired the phenotype of
6-azauracil sensitivity. One is that S-II is essential for the
transcription of another gene that causes 6-azauracil sensitivity, and
the S-II null mutant lacks this gene product. This gene product may not
be essential for the growth of yeast, since the S-II null mutant is not
lethal
(26, 31, 33) . The other possibility is
that many pausing sites are introduced into the yeast genome in the
presence of 6-azauracil, and the S-II null mutant becomes lethal in the
presence of 6-azauracil, as these pausing sites cannot be read-through
by RNA polymerase II in the absence of S-II. With respect to the latter
possibility, Exinger and Lacroute
(45) reported that
6-azauracil inhibits GTP synthesis in S. cerevisiae and also
causes significant decrease in the UTP content. In vitro transcription experiments showed that reduction of at least one of
four nucleoside triphosphates in the reaction mixture induced pause of
transcription elongation and that addition of S-II released this
cessation of transcription elongation
(18, 20, 21, 29) . Therefore, it is
possible that many pausing sites are created in various class II genes
and that their transcriptions are inhibited in the presence of
6-azauracil, interfering with the growth of the S-II null mutant
(46) . If the former possibility is the case, there should be a
specific gene(s) responsible for 6-azauracil sensitivity that is
transcribed only in the presence of S-II. Identification and isolation
of this gene(s) may give a clue to the function of S-II in
vivo. If the latter possibility is correct, this S-II null mutant
should become lethal when the nucleoside triphospate pool in the cells
is reduced by other methods.
It is unlikely that S-II itself has
activity to detoxify 6-azauracil or to disturb the uptake of
6-azauracil, thus making the S-II null mutant sensitive to 6-azauracil,
because, in general, metabolic enzymes are present in the cytoplasm and
barrier proteins are present in the membrane, whereas S-II was found
almost exclusively in the nuclei.
Data were
obtained from the quantitation of the read-through experiments
described in the legend to Fig. 5. Percentage radioactivity of run-off
transcript (RO) or RNA stalled at TIa/total radioactivity (RO +
TIa) was determined by counting the radioactivity in each band in Fig.
5. Each value represents the average of two experiments with S.D.
We thank Dr. Caroline M. Kane for supplying the
template and protocol for arrest-relief assay.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and the product of the PPR2/DST1 gene. The PPR2/DST1 gene was originally identified as the
gene responsible for 6-azauracil sensitivity of yeast
(31) .
STP
catalyzes the transfer of a strand from a duplex linear
molecule of DNA to a complementary circular single strand
(32, 33) , indicating that this protein has pleiotropic
functions. On the other hand, gene disruption experiments revealed that
the S-II null mutant is viable
(26, 31, 33) but
that the mutation induces sensitivity to 6-azauracil
(31) . It
is not known why the S-II null mutant is sensitive to 6-azauracil.
However, 6-azauracil sensitivity is the only prominent phenotype of the
S-II null mutant, and the function of S-II as a transcription factor
may be indispensable for suppressing 6-azauracil sensitivity. To gain
more insight into the structure-function relationship of S-II, we
created various deletion mutants of S-II and examined their abilities
to stimulate RNA polymerase II, relieve arrest, and suppress the
6-azauracil sensitivity of the S-II null mutant. Results indicated that
all of these activities are due to the same motif in the S-II molecule,
suggesting that 6-azauracil sensitivity is caused by loss of function
of S-II as a transcription factor.
S-II Null Mutant TNY14
The S-II null
mutant was established previously from ANY102 ( Mata/Mat,
leu2/leu2, his/his, trp1/trp1, ura3/ura3) by inserting the
LEU2 gene into the S-II gene
(26) . Therefore, the null
mutant required uracil and could not be used for assay of suppression
of 6-azauracil sensitivity. So, we established a new S-II null mutant
by inserting the URA3 gene. Gene disruption method used was
essentially as reported before
(26) . Briefly, pBSM13 carrying a
4.0-kilobase fragment of YSII::LEU2 was digested with
XbaI and ClaI to remove the LEU2 gene, and
the URA3 gene was ligated instead of the LEU2 gene.
The insert was purified and used for transformation of TNY04 obtained
by tetrad dissection of ANY102
(34) . One of the
URA
transformants thus obtained was named
TNY14. Disruption of the S-II gene in TNY14 was confirmed by Southern
blot analysis as described before
(26) .
Yeast Transformation
Yeast cells were
transformed essentially as described by Gietz et al. (34) . They were grown in 5 ml of YPD medium (1%
polypeptone, 1% yeast extract, 2% glucose) at 30 °C to an optical
density at 600 nm ( A) of 2.0 and then collected
and suspended in 50 µl of TELiAc (10 m
M Tris/HCl buffer,
pH 7.5, 1 m
M EDTA, 100 m
M CH
COOLi). This
suspension was incubated with 100 ng of plasmids, 50 µg of
heat-denatured calf thymus DNA, and 300 µl of TELiAc containing 40%
polyethylene glycol 4000 for 30 min at 30 °C and then for 20 min at
42 °C. The cells were collected, suspended in sterilized water, and
spread on YNBD plates (0.67% yeast nitrogen base without amino acids,
2% glucose), supplemented with 20 µg/ml each of adenine sulfate,
tryptophan, histidine, and 30 µg/ml of leucine, and incubated for 3
days at 30 °C.
Assay of Suppression of 6-Azauracil
Sensitivity
Transformed cells were cultured in EMD medium
(0.67% yeast nitrogen base without amino acids, 0.5% casamino acids
technical, 2% glucose) with an appropriate supplement(s) at 30 °C
until Areached about 2.0. Then 2.5
10
cells were transferred to 0.5 ml of fresh medium and
incubated at 30 °C for 2 h. The cell suspension was diluted
1000-fold with sterilized water, and 120 µl of the diluted cell
suspension was spread on YNBD or YNBGS (0.67% yeast nitrogen base
without amino acids, 5% galactose, and 0.2% sucrose) plates containing
an appropriate supplement(s) with or without 100 µg/ml 6-azauracil.
Colonies on YNBGS plates were examined after incubation at 30 °C
for 5 days.
Assay of Stimulation of RNA Polymerase II by S-II
Deletion Mutants
This assay was done essentially as
described before in the presence and absence of each recombinant S-II
deletion mutant protein
(26) . The reaction mixture (60 µl)
contained 50 m
M Tris/HCl (pH 7.9), 1.6 m
M MnCl, 0.5 m
M each of ATP, GTP, and CTP, 0.01
m
M UTP, 18.5 kBq of [
H]UTP, 10 m
M 2-mercaptoethanol, 2 µg of calf thymus DNA, and 8 units of
partially purified S. cerevisiae RNA polymerase II. The
reaction mixture was incubated for 20 min at 30 °C, and then the
radioactivity incorporated into the acid-insoluble fraction was
counted. 1 unit of RNA polymerase II was defined as the amount
incorporating 1 pmol of UMP into the acid-insoluble fraction.
Assay of Arrest-relief Activity of S-II Deletion
Mutants
This assay was done essentially as described by
Christie et al. (29) , using a
3`-deoxycytidine-extended template
(35) of the TaqI
fragment containing the human histone H3.3 gene
(36) , RNA
polymerase II, and S-II mutant proteins. The dC-tailed template used
was kindly provided by Dr. C. M. Kane (University of California,
Berkeley Dept. of Molecular and Cell Biology).
Recombinant S-II Deletion Mutant
Proteins
Recombinant S-II mutant proteins were expressed in
Escherichia coli using the T7 expression system, as described
before
(37) . Plasmids containing S-II deletion mutants were
constructed as follows. S-II deletion mutants were amplified by
polymerase chain reaction using pYSII-2 as a template, and the
resulting DNA fragments were ligated into an expression vector, pET-3d.
The resulting plasmids were transfected into E. coli BL21(DE3)/pLysE, and induction of S-II mutant proteins was
performed by treating the E. coli cells with
isopropyl-1-thio--
D-galactopyranoside.
g for 1 h. The resulting supernatant was diluted with buffer 1 (50
m
M Tris/HCl, pH 7.9, 5 m
M 2-mercaptoethanol, 0.1%
Triton X-100) and then subjected to fast protein liquid chromatography
on a Mono-S HR 5/5 column equilibrated with buffer 1. Recombinant S-II
deletion mutant proteins were eluted with buffer 1 containing
0.05-0.2
M NaCl. The fraction of
3-151
protein from the Mono-S column was further purified on a column of
Superose 12 with buffer 1 containing 1
M NaCl. Recombinant
S-II mutant proteins were detected by immunoblotting with antibody
against yeast S-II.
Other Methods
S-II and partially purified
RNA polymerase II from S. cerevisiae were prepared as
described before
(26) . Antibody against S-II was raised by
injecting purified S-II into male albino rabbits. On immunoblotting,
this antibody detected S-II in the crude extract of yeast as a single
band. DNA manipulations including restriction enzyme digestion, gel
electrophoresis, DNA ligation, plasmid isolation, and E. coli transformation were carried out by standard methods.
SDS-polyacrylamide gel electrophoresis and immunoblot analysis were
performed as described before
(38, 39) . Protein was
determined by the method of Bradford
(40) .
Suppression of 6-Azauracil Sensitivity of the S-II
Null Mutant by S-II Deletion Mutant Proteins in
Vivo
Previous studies showed that S-II, the PPR2/DST1 gene product, and STP are almost certainly the same protein
(26, 31, 33) . We are interested in the
functional motifs in the S-II molecule. To examine whether the region
needed for stimulation of RNA polymerase II and that needed for
6-azauracil sensitivity in the S-II molecule could be separated, we
constructed plasmids carrying the genes for various S-II deletion
mutant proteins and used these plasmids for in vivo and in
vitro experiments. We examined the ability of S-II deletion mutant
proteins to suppress 6-azauracil sensitivity by introducing these
plasmids into the yeast S-II null mutant and expressing the mutant
proteins in vivo. We also expressed these plasmids in E.
coli, isolated mutant S-II proteins, and examined their ability to
stimulate yeast RNA polymerase II in vitro.
Figure 1:
6-Azauracil sensitivity of TNY14 and
its suppression by S-II. To detect suppression of the 6-azauracil
sensitivity of TNY14, pYSG6 carrying the full-length S-II gene was
introduced, and its expression was induced by galactose in the presence
of 6-azauracil. A and B, YNBD agar plates; C and D, YNBGS agar plates containing galactose; A and C contained no drug; B and D contained 100 µg/ml 6-azauracil.
The amino acid
sequence of S-II is known to be conserved in various eukaryotes. In
particular, about 50 residues in the carboxyl-terminal region are
highly conserved (about 70% similarity), and about 80 residues in the
amino-terminal region are also relatively well conserved (about 38%
similarity)
(10, 13, 15, 26, 27, 28) .
Christie et al.
(29) demonstrated that in yeast S-II,
lacking the first 113 residues has the same activity as the full-length
form to read-through the pausing site.
2-123 (in which residues 2-123
were deleted),
2-141, and
3-147 suppressed the
6-azauracil sensitivity of TNY14 but that
2-149 and
2-151 did not. These results indicated that residues
1-147, including the relatively well conserved sequence in the
amino-terminal region, are not essential for suppression of 6-azauracil
sensitivity. The carboxyl-terminal mutant,
260-309, did not
suppress 6-azauracil sensitivity, indicating that the 49
carboxyl-terminal residues are necessary for suppressing 6-azauracil
sensitivity. We confirmed the expression of each deletion mutant
protein in the corresponding transformant by immunoblot analysis, as
shown in Fig. 2.
Stimulation of RNA Polymerase II and Relief of
Transcription Arrest by S-II Deletion Mutant Proteins in
Vitro
We then expressed these S-II deletion mutants in
E. coli, purified each protein to near homogeneity, and
examined its ability to stimulate RNA polymerase II and relieve arrest
in vitro. As shown in Fig. 3 A, each protein gave
essentially a single band on SDS-polyacrylamide gel electrophoresis.
Immunoblotting again showed that all the mutant proteins reacted with
antibody against S-II (Fig. 3 B). As is evident from Fig.
4, wild type S-II and the 2-123 protein had almost the same
stimulatory activity, but the
2-141 protein had less, and
the
2-151 protein had none. Therefore, it is clear that the
first 141 residues are not essential for stimulation of RNA polymerase
II. As the
266-309 protein had no stimulatory activity, the
last 44 residues in the carboxyl-terminal region are essential for
stimulation of RNA polymerase II.
Figure 3:
Purification of various S-II deletion
mutant proteins and their immunoblotting. Recombinant S-II deletion
mutants were expressed in E. coli, and each recombinant
protein was purified. A, SDS-polyacrylamide gel
electrophoresis of recombinant S-II deletion mutant proteins stained
with Coomassie Brilliant Blue (47). B, Immunoblotting of
recombinant S-II deletion mutant proteins by affinity-purified
anti-S-II antibody. Lane 1, intact S-II; lane 2, 2-123 protein; lane 3,
2-141 protein; lane 4,
3-151
protein; lane 5,
266-309
protein.
We also examined arrest-relief
activity of these mutant proteins. As shown in Fig. 5, wild type S-II,
2-123, and
2-141 proteins promoted read-through
by RNA polymerase II at specific blocks to elongation in the human
histone H3.3 gene, sites designated TIa, TIb, and TII
(36, 41) . However,
3-151 and
266-309 proteins had no appreciable activity to relieve
transcription arrest at these sites. Summary of two independent
arrest-relief experiments is shown in Table I. These results strongly
suggested that the ability to stimulate RNA polymerase II and to
relieve arrest at specific pausing sites are the same activity, and
that this activity is essential for suppression of 6-azauracil
sensitivity of S-II null mutants, as summarized in Fig. 6.
2-147 and
2-149 proteins in an
E. coli lysate and thus could not examine their abilities to
stimulate RNA polymerase II and relieve arrest. Nonetheless, it is
quite clear that there is a good correlation between the activity for
suppression of 6-azauracil sensitivity in vivo and that for
stimulation of RNA polymerase II (and thus for arrest relief) in
vitro, and that nearly half of the 309 residues of S-II are not
directly related to these activities. Christie et al. (29) also reported that there is essentially no difference in
the cleavage or read-through activities between complete yeast S-II and
a mutant S-II lacking the first 113 residues. Probably, the functional
motif of S-II is in the carboxyl-terminal half, and the amino-terminal
half is the regulatory motif, since this region of S-II of Ehrlich cell
has been shown to contain phosphorylation sites
(7) . However,
read-through mechanism of S-II may not be so simple because
Cipres-Palacin and Kane
(44) recently reported S-II mutants
that are inactive for promoting read-through, although they stimulated
cleavage of the nascent transcript in stalled elongation complexes
(44) .
260-309 protein had no stimulatory and arrest-relief
activity from the fact that the 6-residue-longer
266-309
protein had no activity.
Table: Summary of activities of S-II mutant
proteins to promote read-through of RNA polymerase II
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.