(Received for publication, November 13, 1995)
From the
Yeast RNA polymerase II enzymes containing single amino acid substitutions in the second largest subunit were analyzed in vitro for elongation-related defects. Mutants were chosen for analysis based on their ability to render yeast cells sensitive to growth on medium containing 6-azauracil. RNA polymerase II purified from three different 6-azauracil-sensitive yeast strains displayed increased arrest at well characterized arrest sites in vitro. The extent of this defect did not correlate with sensitivity to growth in the presence of 6-azauracil. The most severe effect resulted from mutation rpb2-10 (P1018S), which occurs in region H, a domain highly conserved between prokaryotic and eukaryotic RNA polymerases that is associated with nucleotide binding. The average elongation rate of this mutant enzyme is also slower than wild type. We suggest that the slowed elongation rate and an increase in dwell time of elongating pol II leads to rpb2-10's arrest-prone phenotype. This mutant enzyme can respond to SII for transcriptional read-through and carry out SII-activated nascent RNA cleavage.
RNA polymerase II (pol II) ()can encounter a variety
of transcriptional blocks during the elongation phase of RNA synthesis.
These include DNA sequences (reviewed in Kane, 1994; Reines, 1994), DNA
bound proteins (Deuschle et al., 1990; Izban and Luse, 1991;
Kuhn et al., 1990; Reines and Mote, 1993), DNA binding drugs
(Mote et al., 1994), and covalent modifications due to DNA
damage (Donahue et al., 1994). In vitro studies have
identified mechanisms by which elongating pol II can overcome some of
these obstacles. Intrinsic DNA sequences that cause pol II to become
arrested have been most extensively investigated and are frequently
employed as a model in studies of transcriptional arrest.
Stably arrested pol II can be reactivated for RNA chain extension by elongation factor SII. SII binds the enzyme and activates a ribonuclease activity thought to reside within pol II (reviewed in Reines(1994)). This ribonuclease removes a small number of nucleotides from the 3` end of the nascent RNA prior to its re-extension through the blockage (Izban and Luse, 1993a, 1993b; Gu and Reines, 1995b) in a reiterative cleavage and resynthesis process, allowing a pol II molecule multiple attempts at chain extension through an obstacle (Gu et al., 1993; Guo and Price, 1993; Reines, 1994).
The extent of arrest can be reduced or prevented by increasing pol II's rate of nucleotide addition to the nascent RNA chain, which decreases the dwell time of the enzyme at each template position (Bengal et al., 1991; Izban and Luse, 1992; Gu and Reines, 1995a). pol II's elongation rate can also be influenced by ammonium ions (Izban and Luse, 1991, 1992) and elongation factors TFIIF (Flores et al., 1989; Price et al., 1989; Bengal et al., 1991; Izban and Luse, 1992) and SIII (elongin; Aso et al., 1995). Conversely, when nucleotide addition is slowed and dwell time is lengthened by a reduction in nucleoside triphosphate concentration, arrest efficiency increases (Wiest and Hawley, 1990; Wiest et al., 1992; Gu and Reines, 1995a). Similarly, reduced in vitro elongation rate has been correlated with increased termination by other RNA polymerases. ``Slow'' Escherichia coli RNA polymerase mutants are termination-prone (Jin and Gross, 1991), and termination-prone eukaryotic RNA polymerase III mutants containing amino acid substitutions in the second largest subunit are reported to have slowed elongation rates (Shaaban et al., 1995).
Less is known about the presence and nature of transcription elongation obstacles in vivo and of the function of elongation factors in overcoming potential transcriptional blocks. In yeast, disruption of the gene encoding elongation factor SII renders cells sensitive to growth in the presence of 6-azauracil (6AU) (Exinger and Lacroute, 1992; Nakanishi et al., 1992), which causes a 2-3-fold reduction in cellular UTP levels and a 10-fold reduction in GTP levels (Exinger and Lacroute, 1992). Yeast lacking SII are also sensitive to mycophenolic acid, another inhibitor of NTP synthesis (Exinger and Lacroute, 1992).
Mutations of the largest subunit of pol II (RPB1) also confer growth sensitivity to 6AU (Archambault et al., 1992). These mutations are clustered within a 470-base pair region conserved among eukaryotes. These are the only reported mutations in the 12-subunit pol II enzyme that confer 6AU sensitivity. Interestingly, the 6AU sensitivity caused by each of these mutant alleles can be complemented by overexpression of SII (Archambault et al., 1992), providing in vivo evidence for a role of SII in pol II transcription and suggesting that depression of NTP levels increases the reliance of the enzyme upon SII. These findings have also suggested that 6AU sensitivity might serve as a bioassay for mutations in components of the elongation machinery, although a direct demonstration that 6AU-sensitive RPB1 alleles generate an elongation-defective enzyme has not been reported.
Several lines of evidence implicate the second largest subunit of pol II (RPB2) in transcript elongation, including binding of nucleotide substrates (Treich et al., 1992) and RNA products (Bartholomew et al., 1986) during RNA synthesis. Genetic experiments reveal that homology region H, which is strongly conserved among all organisms, is important for the enzyme's function (Scafe et al., 1990a).
The implied role of RPB2 in transcript elongation and potential applicability of 6AU as a screen for mutant alleles that contribute to transcriptional elongation defects prompted us to test the enzymatic activity of 6AU-sensitive RPB2 mutants and assess the relevance of the 6AU-sensitive phenotype with respect to distinct, elongation-related activities in vitro. Here, we report that three mutations in RPB2 that confer 6AU sensitivity to haploid yeast strains display an increased propensity to become arrested at intrinsic DNA sequences in vitro, a trait likely attributable to their reduced rate of factor-independent nucleotide addition.
This protein was
applied to a DEAE 5-PW column (Bio-Rad; 75 7.5 mm). Bound
material was eluted with a 9-ml gradient from 0.1-0.5 M KCl (Conaway and Conaway, 1990). Fractions were assayed by Western
blots using monoclonal antibody 8WG16 (Thompson et al., 1989).
Specific activity of peak fractions was determined in a nonspecific
transcription assay (Sawadogo et al., 1980). Nucleotide
incorporation was greater than 90%
-amanitin-sensitive (100
µg/ml), and specific activity ranged from 18-42 units/mg
protein (1 unit = 1 nmol nucleotide incorporated per minute). In
assays using tailed templates, pol II fractions produced transcripts
that were completely sensitive to
-amanitin. (
)We
estimate a purity of 10-20% by specific activity and silver
staining of SDS gels.
Peak fractions
were pooled and dialyzed in 40 mM Hepes, pH 7.9, 1 mM EDTA, 1 mM DTT, and 0.1 M KCl and applied to a
SP-5PW column (Bio-Rad; 75 7.5 mm). Bound material was eluted
with this buffer and a continuous gradient from 0.1 to 0.55 M KCl (Conaway et al., 1996). The peak fraction of
transcript cleavage and read-through activity (Conaway et al.,
1996) was used in all subsequent experiments. This SII appeared
homogeneous as judged by silver-stained SDS-polyacrylamide gel
electrophoresis.
To facilitate comparison of these assays with those performed
in promoter-driven transcription, we used the transcription conditions
described for a rat liver reconstituted system (Reines et al.,
1987). pol II (0.3 µg of protein) was added to 75 ng of DNA in 20
µl of total volume of 20 mM Hepes, pH 7.9, 20 mM Tris, pH 7.9, 2% (w/v) polyvinylalcohol, 0.4 mg/ml bovine serum
albumin, 12 units of RNasin (Promega), 150 mM KCl, 2 mM DTT. After 30 min at 28 °C, the reaction was diluted using the
same buffer lacking KCl to a final KCl concentration of 60 mM.
After an additional 20 min at 28 °C, MgCl, ATP, GTP and
[
-
P]CTP (3000 Ci/mmol) were added in 6
µl to final concentrations of 7 mM, 20 µM, 20
µM, and 0.6 µM, respectively, resulting in a
transcript 16 nucleotides long, because the first uridine appears at
position +17. Chain extension proceeds in the presence of heparin
(10 µg/ml) and ATP, UTP, GTP, and CTP to synthesize run-off and
arrested RNAs.
As indicated, ternary complexes were immunoprecipitated with anti-RNA monoclonal antibody (Eilat et al., 1982, Reines, 1991). 4 µg of IgG were added per 60 µl of reaction. Complexes were collected after incubation with fixed Staphylococcus aureus cells and centrifugation for 2 min in a microcentrifuge, washed twice in reaction buffer, resuspended in 55 µl, and treated as described in the text. Reactions were stopped by the addition of 0.2 M Tris-HCl, pH 7.5, 25 mM EDTA, 0.3 M NaCl, 2% (w/v) SDS. S. aureus was removed by centrifugation, protein was digested with proteinase K (0.4 mg/ml), and RNA was isolated by ethanol precipitation.
Where indicated, pUC18
was tailed at the SmaI site and cut with PstI as
described, allowing production of a run-off transcript of approximately
2660 nucleotides (Dedrick et al., 1987). Incubations were as
described above; however, RNA was pulse-labeled for 1 min at 21 °C
in the presence of 20 µM each of ATP, UTP, and GTP and 10
µCi of [-
P]CTP (3000 Ci/mmol) per
reaction followed by a chase with 800 µM ATP, UTP, GTP,
and CTP. RNA was subjected to electrophoresis on 7 or 4% polyacrylamide
(19:1, acrylamide:bisacrylamide) gels, which were dried and subjected
to autoradiography and PhosphorImager analysis.
For elongation on pUC18, the rate of accumulation of the maximal level of run-off transcripts (or transcripts 980 nucleotides or larger) was determined by dividing the radioactivity in the run-off length band (or portion of lane containing transcripts of 980 nucleotides or larger) by the average total radioactivity found in the lanes of a gel. To verify that the amount of radiolabeled CTP incorporated into transcripts did not increase with chase time in these reactions, nucleotide incorporation was measured as described (Schwartz et al., 1974). Equal quantities of radioactivity were incorporated at each time point and loaded onto the gels.
Figure 1: Location of mutations in RPB2. The positions of RPB2 mutations considered in this study are indicated. Black boxes show conserved regions of homology between RPB2 and other RNA polymerase polypeptides (see Sweetser et al.(1987) for nomenclature).
Figure 2: 6-Azauracil sensitivity of RPB2 mutants. YPD cultures of each strain were diluted to identical optical densities. 3 µl were streaked onto SD-URA medium and grown for 72 h at 30 °C in the presence or the absence of 6AU (75 µg/ml).
Figure 3:
SII-mediated read-through by arrested
elongation complexes. Arrested ternary complexes were assembled by
extending a 16-nucleotide pulse-labeled transcript for 20 min in the
presence of 800 µM ATP, UTP, and GTP plus 100 µM CTP (lanes 0). Arrested complexes were
immunoprecipitated, washed, and treated with SII (approximately 30-fold
molar excess), MgCl, and 800 µM of ATP, UTP,
GTP, and CTP. Aliquots were stopped at indicated times. A,
wild type; B, rpb2-10; C, rpb2-7; D, rpb2-4. Positions of
arrested (Ia and II) and run-off length transcripts are indicated at
the right of each panel; positions of RNA markers (540, 420,
380, and 260 nucleotides) are indicated by arrowheads on the left. RO, run-off.
Figure 5: Quantitation of read-through of sites Ia and Ib and site II by wild type (solid triangles) and rpb2-10 (open triangles) pol II. Labeled RNAs from gels shown in Fig. 4were quantified using a PhosphorImager as described in the text.
Figure 4: Kinetics of elongation through arrest sites by wild type (A) and rpb2-10 (B) pol II. Transcription reactions were performed as described in the presence or the absence of SII (approximately 150-fold molar excess). Aliquots were stopped at the indicated times. Arrested and run-off length transcripts are identified by the arrows at right. Positions of transcripts are as indicated in the legend to Fig. 3. RO, run-off.
Figure 6: A, effect of CTP concentration on read-through efficiency by wild type (lanes 1-5) and rpb2-10 (lanes 6-10) pol II. Transcription reactions were performed with 800 µM ATP, UTP, and GTP plus the indicated concentration of CTP. Positions of transcripts are as indicated in the legend to Fig. 3. B, quantitation of data in A. Percent read-through was determined using a PhosphorImager as described in the text. Sites Ia and Ib are indicated by closed circles; site II is indicated by open circles. RO, run-off.
Figure 7: A, transcript elongation on pUC18 tailed template. Transcription reactions were performed using wild type (left panel) or rpb2-10 (right panel) pol II, and aliquots were removed and stopped at the indicated times. Position of run-off (RO) transcripts (approximately 2660 nucleotides) is indicated at right. Positions of RNA markers (1164, 540, 420, 380, and 260 nucleotides) are indicated by the arrowheads at left. B, percent of run-off transcripts versus time. Quantitation of data in A for wild type (solid triangles) and rpb2-10 (open triangles) pol II. % run-off = run-off/mean total radioactivity in lane. C, percent transcripts 980 nucleotides or larger versus time. Quantitation of data in A for wild type (solid triangles) and rpb2-10 (open triangles). % > 980 = RNAs 980 nucleotides or larger/average amount radioactivity loaded per lane. The asterisk indicates value significantly different than wild type (p < 0.05 by Student's t test; n = 3)
These relative differences correspond to the different specific activity measurements for each enzyme in traditional nonspecific transcription assays on a poly(rC) template (Ruet et al., 1978). rpb2-10 enzyme had a 2.3-fold lower specific activity than wild type pol II (18 units/mg protein versus 42 units/mg; 1 unit = 1 nmol GTP incorporated per min in a 20-min assay). We also measured activity in an RNA polymerase assay that uses denatured salmon sperm DNA template (Hodo and Blatti, 1977). This assay revealed a 4.3-fold lower specific activity for rpb2-10versus wild type pol II (0.4 versus 1.7 units/mg; 1 unit = 1 pmol CTP incorporated per min in a 20-min assay). Taken together, these three assays suggest that rpb2-10 pol II is generally 2-4-fold slower than the wild type in its average elongation rate.
Figure 8: Nascent RNA cleavage by RPB2 mutant enzymes. Arrested complexes were assembled as described in the legend for Fig. 3. Complexes arrested at site Ia were immunoprecipitated, washed free of NTPs and incubated for 30 min in the absence (lanes 1) or the presence (lanes 2 and 3) of SII (approximately 30-fold molar excess) and 800 µM of ATP, UTP, GTP, and CTP (lanes 3 only). Positions of arrested (Ia) and cleaved (*) transcripts are indicated. A-D show reactions employing wild type, rpb2-4, rpb2-7, and rpb2-10 enzymes, respectively. In E, arrested rpb2-10 complexes were washed free of NTPs (lane 1) and treated with SII and 800 µM of ATP, UTP, GTP, and 3`-dCTP (lane 2). Note: The symbol < in C denotes the boundary between 5 and 15% gel slabs seen only in this panel.
In this study we have identified three mutant alleles of the second largest subunit of yeast pol II, rpb2-4, rpb2-7, and rpb2-10 (Scafe et al., 1990a, 1990b), that confer sensitivity to growth of cells in the presence of 6AU and yield defective pol II enzymes. This is, to our knowledge, the first report of a 6AU-sensitive mutation in pol II that has been shown to yield an altered enzyme activity in vitro.
Presumably, this drug's effect on cellular NTP pools puts a stress on the elongation machinery, resulting in a growth defect (Archambault et al., 1992). This hypothesis seems particularly attractive considering the demonstrated relationship in vitro between arrest efficiency and dwell time (Wiest and Hawley, 1990; Wiest et al., 1992; Gu and Reines, 1995a). A similar relationship has been observed between the rate of nucleotide addition and termination for other RNA polymerases, including slow, termination-prone mutants of E. coli RNA polymerase (Jin and Gross, 1991) and RNA polymerase III (Shaaban et al., 1995). We would expect that depressing the ATP and CTP pools would manifest a growth defect in these and other elongation defective mutants.
These in vitro results are consistent with the in vivo findings relating sensitivity to growth on 6AU with compromised elongation efficiency. The 6AU-sensitive mutations rpb2-4 and rpb2-10 decrease the efficiency with which pol II reads through intrinsic arrest elements in vitro in the absence of SII. The average elongation rate of the rpb2-10 mutant is also reduced. We suggest that the arrest-prone phenotype conferred by this mutation is at least partially due to the slowed overall elongation rate of the mutant pol II molecule.
Although the extent of read-through in the presence of SII is relatively unaffected, the rate at which full-length transcript production is completed is reduced for the rpb2-10 enzyme relative to wild type. This might be expected for a number of reasons. First, more arrested pol II molecules are found at each arrest site for the mutant than the wild type enzyme; so the increased probability of arrest per encounter is likely to mean that on average, more rounds of cleavage and read-through are required for each pol II molecule (Reines, 1994). Second, the presence of tandem arrest sites means a mutant deficiency can manifest itself at, on average, more positions per template than wild type enzyme. Thus, a greater accumulation of enzymes at site II leads to decreased read-through at downstream arrest sites and a higher overall dependence on SII. Finally, the average rate of elongation between arrest sites and between the final arrest site and the end of the template is reduced relative to wild type. That SII enables an arrest-prone mutant pol II to achieve complete read-through is consistent with the rescue of 6AU sensitive pol II alleles by SII overexpression in vivo.
rpb2-10 (P1018S) and rpb2-4 (A1016T) are conditional mutations in homology
region H of yeast pol II's second largest subunit (Scafe et
al., 1990a, b). These residues are very highly conserved across
many species, including representatives of archaebacteria, eubacteria,
fungi, higher plants, many invertebrates, and humans (Iwabe et
al., 1991; Sidow and Thomas, 1994). Two conditional lethal alleles
in the second largest subunit of Drosophila pol II, Z43
(R940H) and M39 (G982E), map in or very near region H (Chen et
al., 1993). Mutation of Lys or Lys
to
Arg in homology region H of Saccharomyces cerevisiae RPB2 is
lethal, and one or both of these residues can be cross-linked to
derivatized nucleotides (Treich et al., 1992). Thus, genetic
and biochemical evidence emphasizes the importance of region H in pol
II function and suggests it is part of a catalytic pocket. If an
alteration in this region affected nucleotide affinity or catalytic
efficiency, slowed reaction speed (i.e. a longer dwell time at
each template position) could result. Reduced reaction rate would mimic
the effect of low substrate concentration, placing the enzyme at
increased risk of becoming arrested when it encounters an arrest signal
in DNA. This predicts that other slow pol IIs, such as that bearing the
-amanitin-resistant C4 mutation in the largest subunit of Drosophila pol II (R741H; Coulter and Greenleaf, 1985), would
also be arrest-prone.
It is important to note that in these in
vitro assays, the read-through ability of mutant pol II enzymes
did not correlate with the relative 6AU sensitivity of the strains
bearing mutant alleles. rpb2-7 pol II reads through
arrest sites with much greater efficiency than rpb2-10.
However, strain Z425, which expresses rpb2-7 pol II, was
the most sensitive to 6AU and mycophenolic acid, whereas
strain Z428, bearing rpb2-10, exhibited a milder 6AU
sensitivity. This suggests that the molecular basis for growth
inhibition by 6AU is complex and may involve components outside the
core enzyme or transcription activities not scored in a promoterless
elongation assay.
The best characterized elongation factors that
affect pol II arrest frequency are TFIIF and SII. We can rule out the
possibility that association of these pol II binding proteins varies
between our preparations of rpb2-7 and wild type enzymes
because the detergent sarcosyl, which inhibits both of these factors,
does not alter the arrest frequencies we measure. As well,
our assay system includes heparin to prevent reinitiation and therefore
is insensitive to the presence of TFIIF (Tan et al., 1995).
Allele-specific extragenic suppression of rpb2-7 might
identify other factors that differentially affect arrest frequency of rpb2-7 pol II to cause 6AU sensitivity in vivo.
pol II's nucleotide binding region, polymerase active site,
nuclease active site, and putative product groove may lie in very close
proximity within the enzyme (Nudler et al., 1995; Wang et
al., 1995). The polymerase and nuclease active sites have been
suggested to be one and the same (Rudd et al., 1994).
Therefore, it is possible that mutations in a single amino acid residue
could cause perturbations affecting more than one biochemical function
of the enzyme. Although it is parsimonious to postulate that reduced
catalytic efficiency of pol II leads to increased arrest and a slowed
response to SII, the rpb2-10 enzyme may have additional
transcriptional defects. Apparent heterogeneity in the lengths of
arrested transcripts, in contrast to the more discrete products
produced by wild type pol II, suggests a heterogeneity in the
recognition of initiation and/or arrest sites. However, primer
extension mapping of the 5` ends of RNA synthesized by these two
enzymes revealed no difference in their start sites on tailed
templates, although it confirmed the heterogeneity of
initiation on tailed templates (Dedrick and Chamberlin, 1985). Arrest
at site Ia by wild type enzyme is intrinsically heterogeneous with RNAs
ending at three consecutive bases (Reines et al., 1987; Gu et al., 1993). Given the unusual heterogeneity of rpb2-10's transcripts, it is difficult to
determine nascent RNA cleavage rates and identify cleavage product
sizes. Use of a promoter-based transcription system to test pol II
activities of these mutants will be important to confirm the elongation
defects, identify other pol II functions that may be impaired in
6AU-sensitive pol IIs, and extend the analysis of the cleavage
reaction.