Analysis of Gene Induction and Arrest Site Transcription in Yeast
with Mutations in the Transcription Elongation Machinery*
Megan
Wind-Rotolo and
Daniel
Reines
From the Graduate Program in Genetics and Molecular Biology and
Department of Biochemistry, Emory University School of Medicine,
Atlanta, Georgia 30322
Received for publication, December 15, 2000
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ABSTRACT |
In vitro, transcript elongation by
RNA polymerase II is impeded by DNA sequences, DNA-bound proteins, and
small ligands. Transcription elongation factor SII (TFIIS) assists RNA
polymerase II to transcribe through these obstacles. There is however,
little direct evidence that SII-responsive arrest sites function in
living cells nor that SII facilitates readthrough in vivo.
Saccharomyces cerevisiae strains lacking elongation factor
SII and/or containing a point mutation in the second largest subunit of
RNA polymerase II, which slows the enzyme's RNA elongation rate, grow
slowly and have defects in mRNA metabolism, particularly in the
presence of nucleotide-depleting drugs. Here we have examined
transcriptional induction in strains lacking SII or containing the slow
polymerase mutation. Both mutants and a combined double mutant were
defective in induction of GAL1 and ENA1. This
was not due to an increase in mRNA degradation and was independent
of any drug treatment, although treatment with the nucleotide-depleting
drug 6-azauracil exacerbated the effect preferentially in the mutants.
These data are consistent with mutants in the Elongator complex, which
show slow inductive responses. When a potent in vitro
arrest site was transcribed in these strains, there was no perceptible
effect upon mRNA accumulation. These data suggest that an
alternative elongation surveillance mechanism exists in
vivo to overcome arrest.
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INTRODUCTION |
Several factors have been identified that act as transcription
elongation factors in vitro. One of the best studied is
transcription elongation factor SII (also known as TFIIS), which
facilitates readthrough of transcription arrest sites and other blocks
to RNA polymerase II (pol
II)1 elongation in
vitro (1, 2). SII acts by binding to pol II and activating an
intrinsic RNA cleavage activity within the enzyme that shortens the
newly transcribed RNA and allows re-extension of the arrested
transcript past the block to elongation (1, 2).
The transcription arrest process has been difficult to study in
vivo, due in part to the rapid processing of primary transcripts and degradation of incomplete transcripts. Sensitivity of yeast to
nucleotide-depleting drugs such as 6-azauracil (6AU) and mycophenolic acid has served as a phenotypic indicator of yeast with mutations in
genes encoding pol II subunits and transcription elongation factors,
including RPB1, RPB2, RPB6,
RPB9, DST1, ELP1, ELP3,
SPT4, SPT5, SPT6, SPT16,
and RTF1 (3-13). This drug sensitivity is thought to result
from stress upon the elongation machinery due to a reduction in the
intracellular pools of nucleotides used for RNA synthesis (3, 8). It is
well known that, in vitro, a low concentration of nucleotide
substrates causes pol II to transcribe at a slower rate, become
arrested more often, and therefore be dependent upon SII for efficient
elongation (4, 14-17). Hence, upon drug treatment, yeast may become
more dependent upon efficient transcript elongation by pol II.
Nevertheless, not all 6AU-sensitive mutants carry mutations in genes
that are obviously related to transcription elongation (18, 19),
underscoring the fact that the mechanistic basis of 6AU sensitivity is
not well understood.
Yeast deleted for ELP1 and ELP3, two genes
encoding Elongator subunits, show delayed transcriptional induction in
response to different environmental stimuli (9, 10). Inactivation of
the elongation-related factor Spt5 when a conditional mutant strain is
shifted to the nonpermissive temperature results in loss of some, but
not all, pol II transcripts (11). In related studies, yeast with a
disruption of the SII gene (dst1) or a point mutation in the
second largest subunit of pol II that reduces elongation rate
(rpb2-10), are sensitive to the nucleotide-depleting drugs
6AU and mycophenolic acid (4, 8, 20). These cells are compromised in
their ability to induce transcription of the gene for IMP
dehydrogenase, IMD2 (also known as PUR5) in
response to 6AU treatment (12). As well, the rpb2-10
mutation results in cold sensitivity and inositol auxotrophy, the
latter phenotype is attributed to the inability to transcribe
INO1 (21). Cells lacking functional SII and containing the
slow elongation mutation display a synergistic sensitivity to 6AU. They
also have reduced poly(A+) mRNA levels and reduced
transcription of a number of genes compared with wild-type (5).
Relatively little is known about the spectrum of genes whose expression
requires or is augmented by elongation factors in general and SII in
particular. Cumulatively, these results suggest that one of the
hallmarks of mutation in elongation factor genes is a slow
transcriptional induction phenotype and a reduction in the efficiency
of mRNA synthesis. Recently, a gene called SSM1
(suppressor of 6-azauracil sensitivity of the SII null mutant 1) was
identified in a high copy suppressor screen of the 6AUs
phenotype of a DST1 deletant (22). SSM1
expression is reduced in DST1 null yeast and is restored by
SII expression (22). Specific sequences in SSM1 that arrest
transcription in vitro could explain why this gene requires
SII for its transcription (22). A pressing question is whether
elongation factors contribute to the efficient transcription of many or
most genes or if they are particularly important for the function of
those genes with specific arrest sites.
Here we have analyzed transcriptional induction in yeast bearing
mutations in the elongation machinery. An advantage of this system is
that the biochemical consequence of this mutation has been
characterized as has the mechanism by which SII relieves arrest
in vitro (2). The effect of 6AU on transcriptional
responses, which is expected to slow elongation rates and promote
arrest in vivo, was also assessed in wild-type and mutant
cells. We find that gene induction is impaired in dst1 and
rpb2-10 single mutants and the rpb2-10 dst1
double mutant in the absence of drug and that this effect is
exacerbated in the presence of 6AU. Finally, we measured the efficiency
of transcription through a strong, well-characterized in
vitro arrest site for yeast pol II in the presence and absence of
6AU. Surprisingly, the arrest site was ineffective in reducing
transcriptional output in any strain in either the presence or absence
of drug. That arrest sites defined in vitro were not
particularly effective in vivo may reflect the fact that
this type of elongation impediment is not rate-limiting in
vivo. SII would therefore seem to participate in gene expression in a somewhat more general manner than would be expected considering the well described arrest-relieving activity of SII in
vitro. An alternative mechanism may exist that prevents arrest in
living cells. Nevertheless, SII and an efficiently elongating pol II have general effects in augmenting transcription in
vivo.
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EXPERIMENTAL PROCEDURES |
Yeast Strains and Growth Medium--
Strains used here are
listed in Table I. Yeast transformation was performed as
previously described (23). The growth medium used for ENA1
induction experiments was synthetic complete medium lacking uracil with
glucose (SC-Ura + Glu), to which NaCl was added to a final
concentration of 1 M. The growth medium for galactose induction experiments was SC-Ura with raffinose (2% w/v; SC-Ura + Raff) (24), to which galactose was added to a final concentration of
2% (w/v). In the glucose repression experiments, glucose was added to
a final concentration of 2% (w/v).
Plasmids--
The plasmids pC1023 and pC1024 were made by
inserting a ~700-base pair fragment (XbaI/NdeI)
containing two tandem Ia arrest sites from the plasmid pAdTerm2-2HH-HH
(Reines et al., 1993) in the correct or inverse orientation,
respectively, into the vector pYES2 (Stratagene, La Jolla, CA).
Northern Analysis--
RNA was prepared by the hot phenol
extraction method and quantitated by measuring absorbance at 260 nm
(25). 15 µg of RNA per sample was run on a 1% agarose-formaldehyde
gel and transferred to a Zeta-Probe GT nylon membrane (Bio-Rad,
Hercules, CA). The filter was dried, baked at 80 °C under vacuum for
30 min, and cross-linked in a Stratalinker 1800 (Stratagene, La Jolla,
CA). Filters were prehybridized for 3 h at 42 °C in 5× SSC,
5× Denhardt's solution (25), 50% formamide, 1% SDS, and 100 µg/ml
salmon sperm DNA. The filters were then hybridized with ~100 ng of
32P-labeled probes overnight at 42 °C, and washed twice
at room temperature in 2× SSC/0.1% SDS for 5 min each, twice at room
temperature in 0.2× SSC/0/1% SDS for 5 min each, and twice at
42 °C in 0.2× SSC/0.1% SDS for 15 min each. Additional washes
(twice at 65 °C in 0.1× SSC/0.1% SDS for 15 min each) were
performed for GAL1, SED1, and pC1023 and pC1024
Northern hybridizations. Filters were exposed to x-ray film and
PhosphorImager screens. Quantitation was performed with a Fujifilm
BAS1000 imaging system. The ENA1 probe was made using
the PCR product amplified using the following primers:
5'-AGGTGCCGTTAACGATATCTGTTCTGA-3' and
5'-CCATCTTAGTAGCAAACACTTGAATCG-3', producing a 400-base pair probe. The
SED1 probe was made using a SmaI/EcoRI
fragment from the SED1 gene. The GAL1 probe was
made using the PCR product amplified from yeast genomic DNA using the following primers: 5'-TCTCGCGAAGAATTCACAAGAGAC-3' and
5'-GCTGCCCAATGCTGGTTTAGAGAC-3', producing a 467-base pair fragment. The
pC1023/pC1024 probe was made using the PCR product amplified from pYES2
using the following primers: 5'-AATAGGGACCTAGACTTCAGG-3' and
5'-CTGCAGATATCCATCACACTG-3', producing a 150-base pair fragment.
The probes were radiolabeled with [
-32P]dATP to a
specific activity of
108 cpm/µg using random hexamer
primers and Klenow DNA polymerase.
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RESULTS |
Induction of ENA1 Transcription Is Impaired in Elongation
Mutants--
Deletion of the Elongator subunit genes ELP1
and ELP3 result in delayed transcriptional induction
responses. To better understand the consequences of mutations that
either slow pol II's elongation rate (rpb2-10) or remove
SII from the cell (dst1), we tested strains bearing either
or both of these changes for their ability to induce transcription of
the sodium-inducible ENA1 gene, which encodes a sodium pump
(26). Cultures were challenged with 1 M NaCl, and RNA was
collected for Northern analysis. A representative experiment is shown
in Fig. 1A, and triplicate
experiments are quantitated in Fig. 1B. Yeast with no SII
(dst1) were slightly defective in their ability to induce
ENA1 transcription relative to wild-type cells, showing a
delay in reaching a maximal level of induction, which was below the
wild-type yeast maxima. Yeast with a slowly elongating pol II
(rpb2-10) had a similar, but more attenuated response than
the dst1 disruptant. Cells with both of these mutations
(rpb2-10 dst1) were quite severely defective in their
ability to induce ENA1 transcription relative to wild-type cells.

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Fig. 1.
Induction of ENA1 in
wild-type and mutant yeast strains. A, RNA from DY103
(WILD TYPE), DY106 (dst1), DY105
(rpb2-10), and DY108 (rpb2-10 dst1) cells were
taken at the time points indicated following addition of NaCl (1 M) to the medium. Blots were probed with a portion of the
ENA1 gene, and results from three separate experiments were
quantitated using a PhosphorImager. ENA1 transcript levels
were corrected for background levels of the same area for each sample
lane. These values were divided by the maximal level of ENA1
transcript present in wild-type at 60 min, and the means and standard
deviations (error bars) were plotted (B).
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Induction of GAL1 Transcription Is Impaired in Elongation
Mutants--
Another well-characterized induction system in yeast is
that of galactose activation of GAL1 transcription. This
response was examined in the elongation-defective strains described
above. Galactose was added to cultures grown in raffinose, and RNA was subjected to Northern analysis. A representative experiment is shown in
Fig. 2A and triplet
experiments were plotted in Fig. 2B. Again we observed that
yeast with mutations in the transcription elongation machinery
(dst1, rpb2-10, and rpb2-10 dst1) were
defective in their ability to induce transcription with respect to both the rate and extent of GAL1 mRNA synthesis. The double
mutant strain (rpb2-10 dst1) was only able to induce
GAL1 mRNA to
37% of the maximal wild-type level
after 3 h of galactose exposure. Although the mutant strains were
not able to attain wild-type levels of either ENA1 or
GAL1 mRNA, they were able to grow on solid medium
containing 1 M NaCl or galactose as their sole carbon source, respectively, suggesting that biologically significant amounts
of gene product were ultimately produced in both cases (data not
shown). Although these cells show reduced levels of total
poly(A)+ mRNA (5), the induction defects shown here
provide more direct evidence of a transcriptional defect in these
cells, which is notable, because it is observed in the absence of any
drug treatment.

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Fig. 2.
Galactose induction of GAL1
in wild-type and mutant yeast strains. A,
Northern analysis was carried out on RNA from DY103 (WILD
TYPE), DY106 (dst1), DY105 (rpb2-10), and
DY108 (rpb2-10 dst1) cells at various times following
galactose addition. Blots were probed for GAL1 mRNA, and
results from three separate experiments were quantitated using a
PhosphorImager. GAL1 transcript levels were corrected for
background levels of the same area for each sample lane. These values
were divided by the maximal level of GAL1 transcript present
in wild-type cells at 120 min, and the means and standard deviations
(error bars) were plotted (B).
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6-Azauracil Treatment Impairs the Ability of Elongation-defective
Yeast to Induce GAL1 Transcription--
6-Azauracil (6AU) is thought
to decrease the elongation rate of transcribing pol II in
vivo because of its ability to inhibit the enzymes that generate
nucleoside triphosphates, thereby producing effects similar to defects
in the elongation machinery (3, 8). To determine whether 6AU would
phenocopy the effect of the rpb2-10 and/or dst1
mutations upon GAL1 induction, yeast were treated with 6AU
for 30 min, galactose was added, and RNA was prepared for Northern
analysis (Fig. 3A). The single
and double mutant strains were significantly impaired in both the rate
and extent of induction in the presence of drug (Fig. 2
versus Fig. 3). In the absence of 6AU, the single mutants
achieved 72% of wild-type GAL1 mRNA levels (Fig.
2B) versus 35-55% in the presence of drug (Fig.
3B). The rpb2-10 dst1 double mutant achieved
maximal induction
37% of wild-type in the absence of drug (Fig.
2B) and 10% in its presence (Fig. 3B). These
results provide additional evidence that 6AU affects transcription, and
it does so in a manner similar to mutations in the elongation
machinery, consistent with the idea that it slows transcription
elongation in vivo subsequent to nucleotide depletion.

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Fig. 3.
The effect of 6AU upon galactose induction of
GAL1 in wild-type and mutant yeast strains.
A, Northern analysis was carried out on RNA from DY103
(WILD TYPE), DY106 (dst1), DY105
(rpb2-10), and DY108(rpb2-10 dst1) cells treated
with 6AU (75 µg/ml) for 30 min before galactose induction. RNA was
prepared at the indicated times, and the blot was probed for
GAL1 mRNA. Results from three separate experiments were
quantitated using a PhosphorImager. GAL1 transcript levels
were corrected for background levels of the same area in each sample
lane. These values were divided by the maximal level of GAL1
transcript in wild-type cells at 120 min, and the means and standard
deviations (error bars) were plotted (B).
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The Loss of GAL1 mRNA Levels after Glucose Shutoff Is Prolonged
in Elongation Mutants--
The reduced amount of GAL1
transcript observed in these mutant strains could be attributed to an
alteration at one of several steps, including the initiation of
transcription of GAL1, elongation through GAL1,
mRNA export, or degradation of the transcript. To test if an
increase in the level of mRNA degradation could account for the
reduced level of GAL1 mRNA in the mutant strains, we
used glucose to repress GAL1 initiation. This repression
mechanism acts by preventing the recruitment of TBP and pol II to the
gene and results in cessation of transcriptional initiation (27-29). GAL1 transcription was induced for 2 h with galactose
then repressed by addition of glucose to growing cultures. RNA was
collected at various times after shut off and its rate of disappearance was analyzed by Northern blotting (Fig.
4A, quantitated in triplicate in Fig. 4B). This experiment shows that the GAL1
transcript is present for a longer period of time after glucose shutoff
in elongation-defective yeast (both single and double mutants) than in
wild-type yeast. The transcript disappeared the least rapidly in the
rpb2-10 dst1 double mutant, somewhat faster in the
rpb2-10 mutant, and the closest to wild-type for the
dst1 disruptant (Fig. 4B). This is not consistent
with the model in which the reduced level of mRNA seen upon
galactose induction in these mutant strains results from an increase in
the rate of transcript degradation (Fig. 2). On the contrary, mRNA
levels disappeared more slowly in the mutant strains with the double
mutant showing the slowest rate of mRNA loss.

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Fig. 4.
Glucose shutoff of GAL1 in
wild-type and mutant yeast. A, Northern blot analysis
was performed on RNA harvested at the indicated times after glucose
addition to cultures of DY103 (WILD TYPE), DY106
(dst1), DY105 (rpb2-10), and DY108 (rpb2-10
dst1) cells grown for 2 h in galactose. RNA was also prepared
from control cells that were not induced with galactose ( ). The blot
was probed for GAL1 mRNA, and results were quantitated
using a PhosphorImager for three separate experiments. GAL1
transcript levels were corrected for background signal in the same area
for each lane. Transcript levels for each strain were calculated
relative to its zero time value, and the means and standard deviations
(error bars) were plotted (B).
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6-Azauracil Treatment Decreases the Rate of GAL1 Transcript
Disappearance--
Because 6AU treatment affected the induction of
GAL1 in a manner similar to that of the mutants, we explored
the possibility that 6AU treatment also had an effect similar to that
of the mutations on the rate of GAL1 transcript
disappearance. Galactose-induced cultures were treated with 75 µg/ml
6AU for 30 min before glucose was added. RNA was prepared for Northern
analysis at times thereafter (Fig. 5).
6AU slowed the rate of transcript disappearance in wild-type cells to a
rate similar to that of untreated double mutant yeast (rpb2-10
dst1). The rate of disappearance of the GAL1 transcript in the double mutant yeast (rpb2-10 dst1) was also slowed
further when treated with 6AU. These data are again consistent with the idea that 6AU phenocopies mutations that perturb transcription elongation and does not operate by increasing the rate of transcript degradation, in further support of the model that delayed
GAL1 induction in the rpb2-10 dst1 mutant yeast
(Fig. 2) is not due to an increase in GAL1 transcript
degradation. The fact that 6AU can make more extreme the defects seen
in the rpb2-10 dst1 strain suggests that the process slowed
by the drug is additive with the effect of mutations in the
transcription elongation pathway.

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Fig. 5.
The effect of 6AU upon glucose shutoff of
GAL1 in wild-type and double mutant (rpb2-10
dst1) yeast. A, Northern blot analysis was
performed on RNA from DY103 (WILD TYPE) and DY108
(rpb2-10 dst1) cells grown for 2 h following addition
of galactose. 6AU (75 µg/ml) was added for 30 min as indicated,
followed by glucose. RNA was prepared at the indicated times as well as
before and after induction by galactose ( gal and + gal lanes). The blot was probed for GAL1 mRNA, and
results were quantitated using a PhosphorImager for three separate
experiments. GAL1 transcript levels were corrected for
background signal in the same area for each lane. The GAL1
transcript levels were expressed as the fraction of starting
GAL1 transcript remaining for each culture, and the means
and standard deviations (error bars) were plotted
(B).
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mRNA Production in Vivo Is Not Influenced by a Strong in Vitro
Arrest Site--
We initially presumed that these elongation mutations
and 6AU treatment operated by depleting nucleotides, slowing
elongation, enhancing arrest, and reducing the ability of pol II to
bypass blocks to elongation, all of which should exacerbate arrest site readthrough in vivo. Presumably, a gene's transcription
level in this case would be related to the number or location of pause or arrest site sequences within it. Until recently, no arrest sites
have been described in yeast gene sequences in vitro nor shown to operate in vivo (22). Hence, we set out to analyze in vivo a well-characterized arrest site (called site Ia;
originally derived from a human histone gene), which is known to
efficiently arrest transcription by yeast and mammalian pol II as well
as phage and bacterial RNA polymerases in vitro (4, 30-35).
High copy yeast plasmids were constructed containing a tandem
arrangement of two Ia arrest sites inserted downstream of the
GAL1 promoter and upstream of the CYC1
polyadenylation/termination signals (pC1023; Fig.
6). Two consecutive Ia sites function
additively in stopping 75% of the polymerase enzymes that encounter
them in vitro (36). Arrest of pol II at the first or second
site would generate 355- and 577-nucleotide transcripts, respectively.
As a control, the tandem Ia sites were inserted in the inverse
orientation, because its arrest function is strictly
orientation-dependent in vitro (pC1024; Fig. 6)
(36). Plasmids pC1023 and pC1024, as well as the empty vector (pYES2),
were individually transformed into wild-type cells and strains with the
dst1 and/or rpb2-10 mutations (Table I). Cells were induced with galactose for
1 h, and RNA was collected for Northern analysis using a probe
that hybridizes downstream of the Ia sequences (Fig. 6). This probe
would detect differences in the total amount of full-length transcript
produced. The mutant strains show lower overall levels of transcript
than wild-type cells, as expected from the induction defect of the
mutants described above (Fig. 2). To facilitate comparison between
replicate Northern blot experiments, we measured the amount of
full-length transcript that contains the arrest sites in the arresting
orientation (pC1023) to that derived from the plasmid with the Ia sites
in the nonarresting (pC1024) orientation. Each of these values were
divided by the amount of transcript produced by the wild-type strain
harboring the vector lacking Ia sites. No significant difference was
seen in the relative ability of any strain to transcribe through the Ia
site in the correct (arresting) versus the inverse
(nonarresting) orientation (Fig.
7A; two-tailed t
test, p > 0.025). In other words, within a strain, the
level of full-length transcript containing the Ia insert in the sense
orientation was similar to that generated from the plasmid with the Ia
array in the inverse orientation (Fig. 7B). Using a probe
complementary to the transcript upstream of the inserted Ia sequences,
we were unable to detect shortened transcripts of the size expected if
pol II became arrested at the Ia sites (data not shown). This indicates
that an in vitro arrest site does not have a strong effect
upon transcription in vivo.

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Fig. 6.
Plasmid constructs used for in
vivo transcription of the Ia arrest site. pC1023 and
pC1024 contain two tandem Ia sites in the sense orientation
(-Ia-Ia ) and the inverse orientation
( Ia-Ia-), respectively. pYES2 is the vector used for
construction of pC1023 and pC1024 and was used as a no-insert control
in these experiments. Primers used for PCR of 5'- and 3'-fragments used
as probes are indicated with the arrowheads.
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Fig. 7.
Transcription of the Ia arrest site in
wild-type and mutant yeast. A, Northern blot analysis
was carried out on RNA prepared from DY500, DY510, DY520 (WILD
TYPE); DY501, DY511, DY521 (dst1); DY502, DY512, DY522
(rpb2-10); and DY503, DY513, DY523 (rpb2-10 dst1)
yeast after 60 min of galactose (+) or no ( ) induction. The blot was
probed with the PCR product 3' of the Ia site, shown in Fig. 6.
Arrows indicate the full-length transcripts produced from
the different plasmids. B, the mean (n = 3)
transcript level for galactose-induced cultures was determined by
PhosphorImager, corrected for background levels, and normalized to
transcript levels in wild-type cells containing pYES2. The means and
standard deviations (error bars) were plotted.
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6AU Does Not Cause pol II to Arrest in Wild-type or
Elongation-defective Yeast--
If a reduction in intracellular NTP
pools increases arrest in vivo as it does in in
vitro, then we reasoned that 6AU treatment of these strains might
reveal an attenuation in transcription at these tandem arrest sites,
particularly in the mutants. Drug-treated cultures were pretreated with
6AU for 30 min. Following drug treatment, cells were induced with
galactose for 1 h, and samples were collected for Northern
analysis. As observed above for galactose induction of the chromosomal
GAL1 gene (Fig. 3), the addition of 6AU decreased the
overall yield of transcript from the plasmids (Fig.
8A). The treatment, however,
did not make detectable an effect of the arrest site on attenuating
transcription when the total amount of full-length transcript produced
from these two plasmids was compared (Fig. 8B; two-tailed
t test, p > 0.025). Similar levels of
full-length transcript were produced when the Ia sites were transcribed
in the arresting orientation versus the nonarresting
orientation in wild-type and dst1 disruptant yeast (Fig.
8B). Levels of full-length transcript in double mutant or
rpb2-10 mutant yeast, were so low they could not be
quantitated. Northern blots were also hybridized with a radiolabeled
probe complementary to a region upstream of the Ia sequences to
determine whether any intermediate-length-arrested transcripts were
produced, and again none were detected in any of the yeast strains
examined (data not shown). Under our assay conditions, 6AU strongly
affects the absolute levels of transcripts produced, but it did not
reveal a role of the Ia site in reducing pol II's ability to elongate
in vivo.

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Fig. 8.
The effect of 6AU upon transcription of the
Ia site in wild-type and mutant yeast. A, Northern blot
analysis was carried out on RNA from DY500, DY510, DY520 (WILD
TYPE); DY501, DY511, DY521 (dst1); DY502, DY512, DY522
(rpb2-10); and DY503, DY513, DY523 (rpb2-10 dst1)
yeast untreated ( ) or treated with 75 µg/ml 6AU (+) for 30 min and
challenged with galactose for 1 h. The blot was probed with the
PCR product 3' of the Ia site, shown in Fig. 6. Arrows
indicate the full-length transcripts produced by the different
plasmids. B, the means and standard deviations
(n = 3) were calculated, normalized to the level of
transcript in untreated wild-type cells containing pYES2, and
plotted.
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DISCUSSION |
The role of eukaryotic elongation factors in facilitating
elongation in vivo is uncertain. In this report we have
examined inducible transcription systems and a synthetic transcription unit containing a known arrest site, to study elongation in living yeast. We provide evidence that mutations in the elongation machinery negatively impact the ability of yeast to carry out two transcriptional induction processes, consistent with prior reports describing the role
of other elongation factors in gene induction and mRNA biosynthesis
in yeast (5, 9-12). Curiously, when transcription of a specific arrest
site known to block elongation in vitro was examined, we did
not find an effect upon RNA production in vivo. This is
surprising, because the DNA sequence used is recognized as a
particularly strong arrest signal that impedes elongation by a number
of RNA polymerases in vitro, including yeast pol II.
Our findings extend to the ENA1 and GAL1
induction systems, recent reports describing a role for SII in the
transcription of the SSM1 gene, the function of which is not
known, and the IMD2 (PUR5) gene (12, 22). The pol
II mutation rpb2-10, which changes a conserved proline near
the active site to a serine and generates an arrest-prone enzyme that
transcribes slowly in vitro, confers a similar
"slow-induction" phenotype upon yeast as do the ELP1,
ELP3, and DST1 deletions. The original
description of this allele included inositol auxotrophy and reduced
levels of INO1 transcription (21). Combining these two
mutations (rpb2-10 and dst1) consistently
resulted in a more severe defect than either alone, in agreement with
earlier findings that the double mutant displayed synthetic 6AU
hypersensitivity, contained less poly(A)+ mRNA, and was
more severely compromised in its ability to induce transcription of
IMD2 (PUR5), than either single mutant (5, 12).
The inefficiency with which the mutant strains induce transcription of
ENA1, GAL1, and IMD2 (PUR5)
strengthens the idea that the efficiency of transcription elongation
contributes to the rate of mRNA synthesis and that SII accelerates
the process in vivo. These data may be the most direct
evidence to date showing that SII augments transcription by pol II
in vivo and is notable in that we observe an influence of
SII and the rpb2-10 mutation on RNA metabolism in the
absence of drug treatment.
Our studies on glucose repression of the GAL1 gene were
prompted by the possibility that the reduction in GAL1
mRNA seen in elongation mutants could be due to enhanced mRNA
degradation. Surprisingly, yeast with both single and double mutations
in their elongation machinery (dst1, rpb2-10, and
rpb2-10 dst1) showed the opposite effect, the
GAL1 transcript took longer to disappear for the double
mutant than either single mutant, in which GAL1 mRNA was
more long-lived than in wild-type cells. This effect was exacerbated in
the presence of 6AU. One interpretation of these findings is that,
subsequent to the repression of new initiation events by glucose, there
may be a residual contribution to the mRNA pool by slowly
transcribing template-engaged pol II enzymes that have yet to reach the
end of the transcription unit. Partially completed primary transcripts
would not be readily observed in Northern assays due to their heterogeneity.
To determine if the mutations in the elongation machinery affect yeast
transcription in a site-specific manner, the ability to transcribe a
known in vitro arrest site was studied in vivo. The effect of transcribing tandem Ia arrest sites was determined, as
well as the effect of 6AU treatment on the transcription of these Ia
sites. Why this arrest site does not attenuate transcription in
vivo is unclear. If the in vitro strength of the tandem
Ia sites was recapitulated in vivo, we would expect
one-fourth of the polymerases to complete synthesis of full-length
transcripts in the absence of SII (dst1). The efficiency of
arrest would be even higher than that in the rpb2-10 dst1
double mutants due to the "hyper-arresting" phenotype of this
polymerase and the cell's lack of SII (4). 6AU treatment of yeast has
been reported to reduce the level of GTP
13-fold in yeast (8) which,
based upon measurements of intracellular GTP concentration, represents a drop of intracellular GTP from 600-1500 µM to 50-115
µM (37). This would bring the GTP concentration to a
level below 200 µM, the Ks of
mammalian pol II for this substrate (38).
By a number of criteria, the defects seen in these yeast strains appear
fairly general, including reduced levels of total poly(A)+
mRNA, reduced levels of a number of individual mRNAs, and the failure of transcriptional induction in at least three systems. All of
these general defects are exacerbated after 6AU treatment (5, 12). The
in vitro data made the relative inactivity of arrest sites
in vivo somewhat surprising. Nevertheless, the in vitro assay may exaggerate the strength of the arrest site
resulting in a more subtle effect in vivo. It is also
possible that a compensating activity present in yeast but not in the
in vitro reaction allows for efficient elongation through
arrest sites. Close homologues of SII are not apparent in the yeast
genome and no eukaryotic proteins other than SII are known that
activate the nascent RNA cleavage reaction employed for arrest site
readthrough. The most closely related gene products are Rpa12, Rpb9,
and Rpc11, subunits of RNA polymerases I, II, and III, respectively.
Rpb9 and Rpc11 have been implicated in transcription elongation (7, 33, 39). pol II, lacking the Rpb9 subunit, is relatively
readthrough-proficient (33). Perhaps the conditional dissociation of
this subunit from elongation complexes can compensate for the absence
of SII in vivo. As well, in the absence of SII, pol II
possesses low but detectable amounts of nascent RNA cleavage activity,
which may suffice to support readthrough in vivo.
Alternatively, other proteins known to increase the average elongation
rate such as TFIIF, Elongator, or elongin may minimize or pre-empt
arrest by helping pol II sustain high elongation rates in
vivo (9, 17, 40, 41). In vitro transcription of arrest
sites by yeast pol II has generally employed promoter-less "tailed"
templates. Activated transcription that takes place from the
GAL1 promoter in vivo may result in a different, more processive form of pol II that may be immune from arrest in
vivo, possibly through modification of pol II at the promoter (42). A synthetic lethal screen has recently revealed activities that
may compensate for the absence of SII in vivo and has drawn a connection between SII and chromatin structure (43). Further work
will be required to test these possibilities.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Cale Lennon for generating some
of the strains and clones used in this study and Drs. Jerry Boss, Sue
Jinks-Robertson, Paul Doetsch, Charlie Moran, and Frank Gordon for
helpful discussions.
 |
FOOTNOTES |
*
This work was supported by Grant GM46331 from the National
Institutes of Health.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: Dept. of Biochemistry,
Emory University School of Medicine, Rollins Research Center, 1510 Clifton Rd., Atlanta, GA 30322. Tel.: 404-727-3361; Fax: 404-727-3452;
E-mail: dreines@emory.edu.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M011322200
 |
ABBREVIATIONS |
The abbreviations used are:
pol II, polymerase
II;
6AU, 6-azauracil;
PCR, polymerase chain reaction.
 |
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