©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Action of -Amanitin during Pyrophosphorolysis and Elongation by RNA Polymerase II (*)

(Received for publication, February 21, 1995; and in revised form, June 7, 1995)

David R. Chafin (§) Hongliang Guo(§) (1) David H. Price (¶)

From theDepartment of Biochemistry, University of Iowa, Iowa City, Iowa 52242 and Experimental Station E400-5435, DuPont/Merck Pharmaceuticals, Wilmington, Delaware 19880

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Using defined elongation complexes formed on dC-tailed templates with Drosophila RNA polymerase II, we have examined elongation, pyrophosphorolysis, and DmS-II-mediated transcript cleavage and the inhibitory effect of alpha-amanitin on these processes. Analysis of pyrophosphorolysis on soluble or immobilized DNA templates confirmed that NTPs are liberated instead of dinucleotides that are released during DmS-II-meditated transcript cleavage. 10 µg/ml alpha-amanitin completely inhibited DmS-II-mediated transcript cleavage but allowed extended pyrophosphorolysis and nucleotide addition to occur. alpha-Amanitin dramatically decreased the V(max) for nucleotide addition but only slightly affected the K for nucleotides. Although the processes are mechanistically distinct, both pyrophosphorolysis and DmS-II-mediated transcript cleavage frequently resulted in similar patterns of shortened transcript. Since polymerase molecules encounter similar kinetic barriers during both processes, it is possible that there is a common step in the reverse movement of the polymerase.


INTRODUCTION

Transcription elongation can be viewed as a repeating cycle of single ribonucleotide incorporation events. Ribonucleotides first bind and then are added to the growing RNA chain, followed by the release of pyrophosphate (PP(i)). The cycle is completed by the downstream translocation of the active site. Early models described extended elongation as a simple repetition of the single cycle and implied monotonic movement of the polymerase(1, 2, 3) . Recent results have demonstrated that the downstream edge of the polymerase can move asynchronously with nucleotide incorporation(4, 5, 6, 7) . This suggests that besides the translocation of the active site that must occur after every nucleotide addition there could be another kind of polymerase movement that occurs during extended elongation (reviewed in (8) ).

In addition to elongation RNA polymerase II can shorten its nascent transcripts in two different reactions. Pyrophosphorolysis is the reversal of elongation utilizing PP(i) as substrate and liberating NTPs. This reaction is dependent on the addition of a divalent cation such as magnesium and proceeds at a much slower rate than elongation(9) . A protein factor S-II causes the polymerase to cleave its nascent transcripts usually in increments greater than mononucleotides reviewed in (10) and (9) and (11) -16. S-II-mediated transcript cleavage is also dependent on the addition of a divalent cation and proceeds rapidly in the presence or absence of NTPs(9, 11, 17) . After S-II-mediated transcript cleavage the polymerase extends the new 3` end and has a second chance of passing the block point(9, 11, 12, 13) .

Although both elongation and S-II-mediated transcript cleavage are sensitive to alpha-amanitin (18, 19) it is not clear how the fungal toxin exerts its effect. Originally, alpha-amanitin was thought to inhibit the formation of phosphodiester bonds(18) . Vaisius and Weiland (20) later showed with calf thymus RNA polymerase II that the first phosphodiester bond could be synthesized, but in this study the formation of additional bonds could not be detected, and it was concluded that the translocation step was blocked. Since relatively short reaction times were used in this study, their results are also consistent with the possibility that alpha-amanitin merely slows the rate of nucleotide incorporation. Other studies have suggested that nucleotide binding is affected by alpha-amanitin(21) . Although it is assumed that pyrophosphorolysis is sensitive to the toxin, this has not been demonstrated.

RNA polymerase from Escherichia coli(22) and RNA polymerase II from yeast (23) and Drosophila(^1)are able to carry out factor-dependent transcript shortening as well as incorporation of nucleotides into RNA chains in the absence of template DNA. When very long reaction times were used both types of reactions with yeast RNA polymerase II were slowed but not completely inhibited by 100 µg/ml alpha-amanitin(23) . Although these results do not allow the determination of the enzymatic step inhibited by alpha-amanitin, they indicate that alpha-amanitin may only affect the rate of the reactions. We show here for the first time that extended pyrophosphorolysis and nucleotide addition can take place in a template-dependent manner in the presence of alpha-amanitin levels previously thought to block the activity of RNA polymerase II.


EXPERIMENTAL PROCEDURES

Materials

Most materials used are the same as used by Guo and Price(9) .

dC-3025 Templates

The soluble dC-3025 template has been described by Price and Parker(24) . Immobilized dC-3025 template was prepared by cloning a 245-base pair fragment, containing the 3` end of the Drosophila H3 histone gene(24) , into the PstI/EcoR I sites of plasmid pSP73 (Promega). Using primers homologous to the SP6 and T7 promoters contained on the pSP73 plasmid, the DNA insert was amplified using 30 cycles of the polymerase chain reaction. The polymerase chain reactions were pooled and loaded onto a 3.5-ml Sephadex G250 column equilibrated with TE, pH 7.5 (10 mM Tris, 1 mM EDTA) and 200 mM NaCl. Fractions containing amplified DNA were digested for 20 min at 37 °C with 200 units of EcoRI. Biotinylation was carried out at room temperature for 25 min and contained 50 µM biotin dATP, 50 mM Tris-Cl, pH 7.5, 5 mM MgCl(2), 1 mM dithiothreitol, 500 µg/ml bovine serum albumin, and 50 units of Klenow fragment in a 100-µl reaction. The biotinylated DNA was digested with PstI and subjected to electrophoresis on a 6% nondenaturing polyacrylamide gel. A gel slice containing the biotinylated 3025 fragment was placed in dialysis tubing, and the DNA was electroeluted into 1 TAE buffer (0.04 M Tris acetate, 0.002 M EDTA) at 90 V for 15 min. The biotinylated 3025 fragment was coupled to streptavidin-conjugated Dyna beads (Dynal Inc.) by incubating 150 µg of washed beads and DNA for 20 min at room temperature. DNA binding was monitored by concentrating the beads and reading the absorbance of the supernatant at 260 nM. The immobilized 3025 fragment was tailed at the PstI site with dCTP and terminal deoxynucleotidal transferase for 5 min at 37 °C. The tailing reaction contained 1 tailing buffer (100 mM cacodylate buffer, pH 6.8, 1 mM CoCl(2), 0.1 mM dithiothreitol), 1 mM dCTP, 50 µg/ml bovine serum albumin, and 40 units of terminal deoxynucleotidal transferase in a 250-µl reaction. The dC-3025 beads were washed 3 times with TE and stored in 150 µl of TE at 4 °C until use.

Formation and Isolation of Elongation Complexes

All reactions were carried out at room temperature (about 21-23 °C). Isolation of IECs (^2)assembled on soluble dC-3025 template using Drosophila RNA polymerase II was as described by Guo and Price(9) . Reactions containing immobilized dC-3025 template were carried out as follows. In a typical 15-µl preincubation Drosophila RNA polymerase II (0.44 units/reaction) was incubated with dC-3025 beads (13.3 ng/reaction) for 10 min in the presence of 25 mM HEPES pH 7.6, 5 mM MgCl(2), 60 mM KCl, and 2 mg/ml bovine serum albumin. Short transcripts were generated (8 nucleotides in length) by adding 35 µl of a solution containing 25 mM HEPES pH 7.6, 5 mM MgCl(2), 0.6 mM GTP and ATP, 75 mM NH(4)Cl, and 100 µCi of [alpha-P]CTP for 30 s. The beads were concentrated and washed 3 times with 200 µl of HMKB (25 mM HEPES pH 7.6, 5 mM MgCl(2), 60 mM KCl, and 200 µg/ml bovine serum albumin). Short transcripts were elongated further by resuspending the beads in 50 µl of a solution containing 25 mM HEPES, 5 mM MgCl(2), 60 mM KCl, and 0.6 mM NTPs for 8 min. Final transcription complexes (IECs) were washed as before with HMKB. IECs were resuspended in HMKB.

Pyrophosphorolysis

IECs were incubated with the indicated concentration of sodium pyrophosphate for different incubation times in the presence of 5 mM MgCl(2). Where indicated, alpha-amanitin was added to the complexes after the MgCl(2) for at least 2 min before other factors were added. Reactions were then stopped with 200 µl of Sarkosyl solution (1% Sarkosyl, 100 mM NaCl, 100 mM Tris, pH 8.0, 10 mM EDTA, and 200 µg/ml tRNA). The nucleic acids were isolated by phenol extraction and ethanol precipitation as described by Sluder et al.(25) and were analyzed in 18% polyacrylamide, 6 M urea, TBE gels as indicated. The gels were dried and exposed to Kodak x-ray films.

Analysis of Pyrophosphorolysis Products

IECs were split into two equal parts. One half was incubated with 1 mM sodium pyrophosphate for 30 min, and the other half was used as a control. Samples were loaded on a 1-ml Mono Q column for oligonucleotide separation as detailed in Guo and Price(9) .

K(m)and VDeterminations

Autoradiographs in Fig.7were scanned with a GS-670 densitometer at 100-µm resolution. The disappearance of the 5-mer was quantitated, and the background was subtracted from the raw values. The percentage 5-mer remaining was calculated by comparing the intensities of individual 5-mer bands to the intensity of the minus nucleotide 5-mer band for each gel. Elongation rates were then determined by subtracting the percentage of 5-mer remaining from 100% and dividing by the total time of the reaction. A reciprocal plot of 1/nucleotides added per second versus 1/substrate concentration was done for the points at which changes in the amount of 5-mer could be most accurately determined. The Kvalue was read directly from the reciprocal plot as -1/x-intercept, and the V(max) was 1/y-intercept.


Figure 7: NTP addition to PP(i)-treated complexes. IECs formed on immobilized dC-3025 with Drosophila RNA polymerase II were isolated as described under ``Experimental Procedures.'' Complexes were treated with 1 mM PP(i) for 90 min and then washed. Autoradiographs: panelA, indicated amounts of NTPs were added to complexes for 6 s; panelB, complexes were treated with 10 µg/ml alpha-amanitin 2 min prior to the addition of the indicated amounts of NTPs for 30 min. Transcripts were analyzed as described in the legend to Fig.1. K and V(max) values were determined by plotting 1/activity versus 1/[substrate] for each reaction (see ``Experimental Procedures'').




Figure 1: Pyrophosphorolysis of isolated elongation complexes. IECs formed on soluble dC-3025 with Drosophila RNA polymerase II were isolated as described under ``Experimental Procedures.'' The IECs were incubated with the indicated concentrations of PP(i) or DmS-II for the indicated times. Nucleic acids were analyzed on an 18% polyacrylamide (60:1 acrylamide/bisacrylamide), TBE, urea gel. Transcript sizes are indicated in nucleotides.




RESULTS

The Transcript Pattern Generated by Pyrophosphorolysis Is Similar to That Generated during DmS-II-mediated Transcript Shortening

We first wanted to demonstrate that IECs were active for pyrophosphorolysis. IECs formed on a soluble dC-tailed template were separated from free nucleotides and free proteins by chromatography on Sephacryl S400 as described under ``Experimental Procedures.'' Fig.1shows an autoradiograph of a gel in which labeled transcripts were analyzed. The IECs used for this experiment contained nascent transcripts predominately 13 nucleotides in length (Fig.1, lane1). When the IECs were incubated with 1 mM sodium PP(i) in the absence of NTPs for 0.3-90 min, the 13-mer, as well as the small amount of 12-mer, gradually disappeared, and new transcripts were generated with higher mobilities (Fig.1, lanes2-7). The appearance of these shortened transcripts was dependent on the presence of pyrophosphate (Fig.1, lane15). The first detectable product was a transcript 11 nucleotides in length, followed by the 9-mer, 7-mer, and 5-mer. When the complexes were incubated with increasing concentrations of pyrophosphate a similar pattern was obtained (Fig.1, lanes8-12). The pattern generated by pyrophosphorolysis was very similar to that generated during DmS-II-mediated transcript shortening (Fig.1, lanes13 and 14). The main difference between the pattern of transcripts generated by pyrophosphorolysis and DmS-II-mediated transcript cleavage was that the small amount of 12-mer seems to be shortened to produce 10-mer and subsequently 8-mer in the presence of DmS-II. Neither the 10-mer nor the 8-mer were detected during pyrophosphorolysis.

There are two possible explanations for the transcript pattern generated during pyrophosphorolysis. PP(i) could mimic the function of DmS-II, causing the liberation of nucleotide dimers(9) . Another explanation is that PP(i) might remove one nucleotide at a time with the apparent 2-nucleotide increment being caused by a barrier related to the removal of nucleotides in that sequence. To resolve this issue the labeled products generated during a 30-min pyrophosphorolysis reaction were combined with unlabeled RNA standards and analyzed by chromatography on Mono Q. Fig.2shows that the small products generated during pyrophosphorolysis were exclusively nucleoside triphosphates that elute between nucleotide trimers and tetramers(9) . Therefore, the 2-nucleotide increment reflects the existence of a barrier to extended pyrophosphorolysis that occurs every 2 nucleotides on this template.


Figure 2: Analysis of the products generated during pyrophosphorolysis. IECs formed on soluble dC-3025 ([alpha-P]CTP-labeled) were incubated with 1 mM sodium PP(i) for 30 min. A control reaction containing an equal amount of complexes was incubated with water for the same time. The radioactive products generated were analyzed using a Mono Q column as described under ``Experimental Procedures.'' Nucleotide monomer, dimer, etc. are indicated by numbers above the elution profile.



alpha-Amanitin Inhibits Pyrophosphorolysis and DmS-II-mediated Transcript Shortening Differently

Although the pattern of transcripts generated during pyrophosphorolysis and DmS-II-mediated transcript shortening are similar, we wanted to determine if alpha-amanitin would inhibit the same mechanistic step. Inhibition of RNA polymerase II activity by alpha-amanitin is a characteristic feature of the enzyme(26, 27) ; however, very limited nucleotide addition in the presence of alpha-amanitin has been observed(20) . Pyrophosphorolysis was slightly inhibited at low levels of alpha-amanitin (Fig.3, compare lanes2 and 3). Most of the transcripts accumulated at the first reverse barrier, the 11-mer, but some of the transcripts were able to be further shortened. At higher levels of alpha-amanitin transcripts accumulated exclusively at the first reverse barrier in the time of the reaction (Fig.3, lanes4-7). The inhibition of pyrophosphorolysis is consistent with the published K for inhibition of RNA synthesis of about 0.05 µg/ml(18) . Addition of a 1,000-fold higher concentration of alpha-amanitin did not suppress the appearance of the 11-mer.


Figure 3: Inhibition of pyrophosphorolysis and DmS-II-mediated transcript shortening by alpha-amanitin. IECs formed on soluble dC-3025 were treated with the indicated amounts of alpha-amanitin for 5 min before the addition of any other factor. The alpha-amanitin-treated complexes were then incubated with 1 mM sodium PP(i) or 40 nM DmS-II for 30 min. In lane14 alpha-amanitin-treated complexes were incubated with 600 µM of all four nucleotides for 30 min. Transcripts were analyzed as described in the legend to Fig.1.



In comparison with pyrophosphorolysis, DmS-II-mediated transcript cleavage responded differently to increasing concentrations of alpha-amanitin (Fig.3, lanes8-14). At low concentrations of alpha-amanitin some complexes were able to carry out multiple rounds of transcript cleavage resulting in the production of the 5-mer. At higher concentrations of alpha-amanitin nearly all of the complexes were blocked for transcript cleavage. The small percentage of ternary complexes that could undergo transcript shortening even when 10 µg/ml of alpha-amanitin was used could be due to competition between DmS-II and alpha-amanitin for interaction with the polymerase. This competition also emerged when the DmS-II concentration was titrated from 4 to 400 nM with a constant 0.5 µg/ml alpha-amanitin concentration (data not shown).

These results suggest that when alpha-amanitin is bound to the polymerase at least one nucleotide can be removed from the nascent transcript in the presence of PP(i). Due to a significant amount of 12-mer in the starting material (Fig.3, lane1) it was not possible to determine if more than one nucleotide could be removed in the presence of alpha-amanitin. The differential sensitivity of pyrophosphorolysis and DmS-II-mediated transcript shortening to alpha-amanitin again suggests that the two processes utilize different mechanisms.

The ability of the polymerase to carry out limited pyrophosphorolysis in the presence of high concentrations of alpha-amanitin demonstrates that the active site is not rendered inactive by the toxin. This is further supported by the reduction of the 12-mer after readdition of all four NTPs to the complexes that were incubated with alpha-amanitin (Fig.3, compare lane14 with lane1). This observation reveals that nucleotide addition to the 12-mer is not completely inhibited by alpha-amanitin. A possible explanation for these observed results is that alpha-amanitin does not stop the catalytic activity of the active center but rather slows the overall rate of nucleotide addition or removal.

alpha-Amanitin Slows but Does Not Block Extended Pyrophosphorolysis

In order to determine if extended pyrophosphorolysis can take place in the presence of alpha-amanitin, IECs that contained a high percentage of 13-mer and low levels of 12-mer were formed and isolated on an immobilized dC-3025 template (see ``Experimental Procedures'' and Fig.4, lane 1). The use of an immobilized template allowed rapid isolation of elongation complexes and more precise manipulation of reaction conditions.


Figure 4: Extended pyrophosphorolysis in the presence of alpha-amanitin. IECs formed on immobilized dC-3025 with Drosophila RNA polymerase II were isolated as described under ``Experimental Procedures'' and treated with 10 µg/ml alpha-amanitin 2 min before the addition of PP(i). The alpha-amanitin-treated complexes were then incubated with 1 mM PP(i) for the indicated times. In lanes2 and 3 complexes were incubated with 600 µM of all four nucleotides for 90 min. Transcripts were analyzed as described in the legend to Fig.1.



When NTPs were added back to IECs for 90 min, an increase in runoff was obtained as well as a reduction in the amount of 13-mer and an intermediate length transcript (Fig.4, lane2, arrow). In the presence of alpha-amanitin nucleotide addition was not observed (Fig.4, lane3). When 1 mM PP(i) was added to the complexes for increasing times the appearance of 11-mer, 9-mer, 7-mer, and 5-mer was similar to that obtained on the soluble dC-3025 template (Fig.4, lanes4-7). In the presence of 10 µg/ml alpha-amanitin extended pyrophosphorolysis still occurred (Fig.4, lanes8-11). At the longest time point most of the complexes reached the first reverse barrier (11-mer); however, a small but significant number of complexes reached the second and third reverse barriers (9-mer and 7-mer). These results are consistent with pyrophosphorolysis being slowed but not blocked by alpha-amanitin. The autoradiograph was scanned, and the disappearance of the 13-mer was quantitated. The disappearance rate of the 13-mer in the presence of alpha-amanitin was 22 percent of the rate in the absence of alpha-amanitin. The appearance of the 9-mer and 7-mer at the 90-min time points is consistent with a similar decrease in the rate of pyrophosphorolysis at each reverse barrier. It was not possible to determine the rate of pyrophosphorolysis at sites between the reverse barriers because transcripts of these lengths (10, 8, etc.) were not detectable.

Similar results are seen with an intermediate length transcript (Fig.4, arrow). This transcript was able to be efficiently elongated upon addition of NTPs unless alpha-amanitin was present (Fig.4, lanes2 and 3). A variety of shorter transcript sizes arose from this transcript upon addition of PP(i) to the reaction (Fig.4, lanes4-7). At least one of the shorter transcripts appeared in the presence of alpha-amanitin (lanes8-11). Two important conclusions can be drawn from these results. The first is that alpha-amanitin slows down but does not completely inhibit the catalytic activity of the active site of the polymerase. The second is that extended pyrophosphorolysis can take place in the presence of alpha-amanitin levels known to block elongation from forward pause sites.

Nascent Transcripts Can Be Extended from PP(i)-treated Complexes

Since transcripts could be shortened in the presence of alpha-amanitin we wanted to determine if transcripts in PP(i)-treated complexes could be re-elongated. IECs formed on immobilized dC-3025 template were treated with 10 µg/ml alpha-amanitin and 1 mM PP(i) for 90 min, washed, and then incubated with NTPs for increasing times (Fig.5, timeline). PP(i)-treated complexes were predominately backed up to the first reverse barrier at nucleotide 11 in 90 min (Fig.5, lane1). Addition of NTPs to these complexes allowed the resynthesis of the 13-mer in the presence or absence of additional alpha-amanitin (Fig.5, lanes2-9). The rate of nucleotide addition was the same whether or not additional alpha-amanitin was included in the final elongation, indicating that the toxin remained tightly bound to the polymerase. The rate of transcript elongation was considerably slower than elongation in the absence of any alpha-amanitin, again suggesting that the catalytic activity of the polymerase is not completely inhibited but rather that the overall reaction is slowed. As was seen earlier alpha-amanitin inhibits the progression of elongation complexes through forward pause sites. One possibility is that polymerases at pause sites require a significant time to continue elongation, and nucleotide addition is furthered slowed by alpha-amanitin. It appears that each nucleotide addition is slowed as is evidenced by the appearance of 12-mer as a major block to elongation.


Figure 5: Nucleotide addition to alpha-amanitin-treated complexes. IECs formed on immobilized dC-3025 with Drosophila RNA polymerase II were isolated as described under ``Experimental Procedures.'' 10 µg/ml alpha-amanitin was added 2 min prior to a 90-min incubation with 1 mM PP(i). Complexes containing shortened transcripts were then washed and incubated again with alpha-amanitin for 2 min as indicated. alpha-Amanitin-treated complexes were incubated with 600 µM of all four nucleotides for the indicated times (see reactionline). Transcripts were analyzed as described in the legend to Fig.1.



In order to determine if more extensive elongation could occur in the presence of alpha-amanitin, IECs containing mainly the 13-mer were formed and then treated with 1 mM PP(i) for 90 min. After washing, the resulting complexes contained transcripts from 5 to 9 nucleotides in length (Fig.6, lane 1). When NTPs were added back in the absence of alpha-amanitin to these complexes, transcripts were elongated to nucleotide 13 in less than 10 min (Fig.6, lanes2-6). In the presence of 10 µg/ml alpha-amanitin transcript elongation was slowed but not blocked (Fig.6, lanes7-11). After 90 min in the presence of alpha-amanitin, some but not all of the polymerase molecules were able to synthesize transcripts 13 nucleotides in length. alpha-Amanitin appeared to slow the addition of every nucleotide as evidenced by an accumulation of transcripts at bands corresponding to nucleotide positions 8, 9, 10, 11, and 12. Similar results are seen with a longer transcript (Fig.6, arrow). This transcript was generated by shortening the long transcript previously described (see Fig.4, arrow). Addition of NTPs to complexes containing this shortened transcript allowed re-elongation to the original forward pause site (Fig.6, lane2). Further incubation resulted in more runoff transcripts and movement through the original pause site (Fig.6, lanes2-6). In the presence of alpha-amanitin, transcripts were able to add nucleotides back to, but not through, the original pause site in 90 min (Fig.6, lanes7-11).


Figure 6: Extended nucleotide addition in the presence of alpha-amanitin. IECs were treated with 1 mM PP(i) for 90 min and then washed. Where indicated, complexes were treated with 10 µg/ml alpha-amanitin 2 min prior to NTP addition. Complexes containing shortened transcripts were incubated with 600 µM of all four nucleotides for the indicated time. Transcripts were analyzed as described in the legend to Fig.1.



alpha-Amanitin Reduces V(max)but Not Kfor a Single Nucleotide Addition

In order to examine the cause of the slower reaction in the presence of alpha-amanitin K and V(max) values were determined for the addition of ATP to transcripts 5 nucleotides long in the presence or absence of 10 µg/ml alpha-amanitin (Fig.7). IECs were treated with 1 mM PP(i) for 90 min, washed, and incubated with or without alpha-amanitin. Complexes were then treated with increasing concentrations of NTPs for 6 s in the absence of the inhibitor or for 30 min in the presence of the inhibitor. In the absence of alpha-amanitin, elongation was not detected at nucleotide concentrations below 1 µM. The largest change in reaction rate was detected at nucleotide concentrations between 3 µM and 100 µM, and adding higher concentrations had minimal effects (Fig.7A, autoradiograph). In order to determine single nucleotide addition rates the disappearance of the 5-mer was quantitated. Elongation rates were defined as the amount of 5-mer that had disappeared divided by the total reaction time (see ``Experimental Procedures'' for details). In order to determine Kand V(max) values, 1/(nucleotides/s) versus 1/[NTP] was plotted for the points that displayed a linear response (Fig.7A, graph). In the absence of alpha-amanitin, RNA polymerase II had a K and V(max) of 9.7 µM and 18.7 nucleotides/s, respectively. In the presence of alpha-amanitin elongation was first observed when the nucleotide concentration reached 300 nM, and the rate increased until approximately 10-30 µM, at which point adding higher concentrations did not affect the rate significantly (Fig.7B, autoradiograph). Nucleotides became inhibitory for elongation in the presence of alpha-amanitin when the concentration reached 1000 µM. In the presence of alpha-amanitin K and V(max) values were 32.8 µM and 0.3 nucleotides/s respectively. alpha-Amanitin slowed the addition of ATP to transcripts 5 nucleotides long to 1.6% of the rate seen without alpha-amanitin. The determined K value did not change nearly as dramatically in the presence of alpha-amanitin. Since nucleotide binding has a major influence in determining the K for elongation by RNA polymerase II, it is very unlikely that that changes in nucleotide binding can explain the slower elongation rate.

It is also possible that extended nucleotide addition in the presence of alpha-amanitin is due to slippage of the RNA through the active site. We do not think this is the case, since PP(i)-treated complexes incubated first with alpha-amanitin and then with pairs of nucleotides for 90 min were able to carry out only the appropriate elongation (data not shown).


DISCUSSION

We have examined the properties of RNA polymerase II during elongation, pyrophosphorolysis, and DmS-II-mediated transcript cleavage and have investigated the inhibitory effect of alpha-amanitin on these processes. Our results show that extended nucleotide addition and pyrophosphorolysis can take place in the presence of alpha-amanitin, which was previously thought to completely block these processes. Our results also suggest that although there are similarities in the pattern of transcripts generated during extended pyrophosphorolysis and DmS-II-mediated transcript cleavage, the two processes are mechanistically distinct.

alpha-Amanitin Slows but Does Not Block Elongation or Pyrophosphorolysis

alpha-Amanitin has been used as an inhibitor of RNA polymerase II for over 25 years(28) , but details of its mechanism of action are unknown. It binds tightly to RNA polymerase II with 1:1 stoichiometry but does not influence the enzyme's affinity for nucleotides(18) , nor does it disrupt assembled elongation complexes (25) . Early studies led to the conclusion that alpha-amanitin inhibits phosphodiester bond formation(18) . Other studies demonstrated the synthesis of one phosphodiester bond in the presence of alpha-amanitin (20) . Our results have shown for the first time that multiple nucleotides can be added or removed by RNA polymerase II in an elongation complex in the presence of alpha-amanitin. It is possible that extended elongation in the presence of alpha-amanitin was not seen in previous studies because elongation times may not have been long enough to observe the slow reaction. In addition, using any reasonable reaction times, we have not observed elongation through pause sites in the presence of amanitin. We found a greater reduction in the rate of elongation than in the rate of pyrophosphorolysis.

Since in the absence of alpha-amanitin the rate of pyrophosphorolysis is much slower than that of elongation, the rates of the two processes are similar in the presence of alpha-amanitin. Since the rate of the incorporation of a single nucleotide is dramatically slowed by alpha-amanitin, the simplest interpretation is that phosphodiester bond formation is slowed. This interpretation is consistent with the lack of a significant change in K, which is influenced by nucleotide binding and the ability to add multiple nucleotides (translocation) in the presence of alpha-amanitin. It is possible that amanitin slows the translocation rate, but this would mean that in our studies complexes containing the 5-mer would need to translocate before nucleotide 6 was added. It is also possible that amanitin could slow the rate of the conformational change that occurs after nucleotide binding found with DNA polymerases(29) .

Pyrophosphorolysis and DmS-II-mediated Transcript Shortening Are Mechanistically Distinct but May Share Common Steps

There are a number of differences between pyrophosphorolysis and DmS-II-mediated transcript cleavage. First, the reactions catalyzed are different, pyrophosphorolysis versus hydrolysis. Although there are potential similarities in the reaction mechanisms, the substrates are radically different in size and charge. Another difference is that pyrophosphorolysis generates NTPs, while DmS-II-mediated transcript cleavage yields predominantly nucleotide dimers of the type pNpN and some nucleoside monophosphates(9, 12) . Also, we showed here that the effect of alpha-amanitin on both processes was different. Pyrophosphorolysis was merely slowed by the toxin, while DmS-II-mediated transcript cleavage was blocked.

While the two processes have significant differences, they may share a common step. The pattern of transcripts generated during pyrophosphorolysis and DmS-II-mediated transcript shortening are similar using dC-3025. Other eucaryotic enzymes and E. coli RNA polymerase give similar results(30, 31) . During extended reactions both processes require that the polymerase move in a reverse direction on the template. If there are kinetic barriers to such reverse movement both processes could be slowed at the same sites even though the reactions driving the reverse movement are different. This idea is supported by the data presented here, which show that similar kinetic barriers are found during both processes. One possibility is that barriers to the reverse movement are related to the sites of discontinuous movement seen during elongation by E. coli polymerase (32, 33) and RNA polymerase II(6) . Inability to extend the downstream contacts of the polymerase with the template can cause pausing in the forward direction(32) . It is possible that inability to release downstream contacts during reverse movement could cause similar barriers during extended pyrophosphorolysis or DmS-II-mediated transcript cleavage. Reverse barriers of this sort might be expected to occur at sites that are normally poised for elongation. Consistent with this we have observed that reverse barriers never coincide with forward pause sites.

A major question that remains to be answered is whether the catalytic center that carries out elongation and pyrophosphorolysis is also responsible for DmS-II-mediated transcript cleavage. If one center carries out both reactions it must have a substrate binding site that can change from binding the highly charged PP(i) molecule to binding water when S-II is present. We showed here that pyrophosphorolysis and DmS-II-mediated transcript cleavage are affected differently by alpha-amanitin. While this could be taken as evidence for two sites it is possible that a single active center catalyzes both reactions, and the differential effect of alpha-amanitin could be due to blockage of DmS-II binding by the toxin. Unfortunately, it is difficult to determine whether alpha-amanitin inhibits the binding of DmS-II since this protein does not bind Drosophila RNA polymerase II tightly. Recently, it was shown that PP(i) was able to cause RNA polymerase II to cleave its nascent transcript endonucleolytically, releasing large nucleotide products, similar to the cleavage pattern by S-II(30) . These data were taken as evidence for the existence of only one active site. Circumstantial evidence for two active centers include: 1) a domain in the large subunit of RNA polymerase II with sequence similarity to a nuclease(34) , and 2) the existence of both an elongation site and a hydrolysis site in functionally similar DNA polymerases. Resolution of the problem may await genetic evidence for the separation of the two functions in RNA polymerase.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM35500. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These two authors contributed equally to this work.

To whom correspondence should be addressed.

^1
D. R. Chafin, H. Guo, and D. H. Price, unpublished results.

^2
The abbreviation used is: IEC, isolated elongation complex.


REFERENCES

  1. Krakow, J. S. and Fronk, E. (1969) J. Biol. Chem. 244,5988-5993 [Abstract/Free Full Text]
  2. Gamper, H. B., and Hearst, J. E. (1982) Cell 29,81-90 [Medline] [Order article via Infotrieve]
  3. von Hippel, P. H., Bear, D. G., Morgan, W. D., and McSwiggen, J. A. (1984) Annu. Rev. Biochem. 53,389-446 [CrossRef][Medline] [Order article via Infotrieve]
  4. Krummel, B., and Chamberlin, M. J. (1989) Biochemistry 28,7829-7842 [Medline] [Order article via Infotrieve]
  5. Linn, S. C., and Luse, D. S. (1991) Mol. Cell. Biol. 11,1508-1522 [Medline] [Order article via Infotrieve]
  6. Rice, G. A., Kane, C. M., and Chamberlin, M. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,4245-4249 [Abstract]
  7. Krummel, B., and Chamberlin, M. J. (1992) J. Mol. Biol. 225,239-250 [Medline] [Order article via Infotrieve]
  8. Chan, C. L., and Landick, R. (1994) in Transcription: Mechanisms and Regulation (Conaway, R. C., and Conaway, J. W., eds) pp. 297-321, Raven Press, Ltd., New York
  9. Guo, H., and Price, D. H. (1993) J. Biol. Chem. 268,18762-18770 [Abstract/Free Full Text]
  10. Kassavetis, G. A., and Geiduschek, E. P. (1993) Science 259,944-945 [Medline] [Order article via Infotrieve]
  11. Izban, M. G., and Luse, D. S. (1992) Genes & Dev. 6,1342-1356
  12. Izban, M. G., and Luse, D. S. (1993) J. Biol. Chem. 268,12864-12873 [Abstract/Free Full Text]
  13. Izban, M. G., and Luse, D. S. (1993) J. Biol. Chem. 268,12874-12885 [Abstract/Free Full Text]
  14. Reines, D., Ghanouni, P., Li, Q., and Mote, J., Jr. (1992) J. Biol. Chem. 267,15516-15522 [Abstract/Free Full Text]
  15. Gu, W., Powell, W., Mote, J., Jr., and Reines, D. (1993) J. Biol. Chem. 268,25604-25616 [Abstract/Free Full Text]
  16. Wang, D., and Hawley, D. K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,843-847 [Abstract]
  17. Reines, D. (1992) J. Biol. Chem. 267,3795-3800 [Abstract/Free Full Text]
  18. Cochet-Meilhac, M., and Chambon, P. (1974) Biochim. Biophys. Acta 353,160-184 [Medline] [Order article via Infotrieve]
  19. Reines, D., Chamberlin, M. J., and Kane, C. M. (1989) J. Biol. Chem. 264,10799-10809 [Abstract/Free Full Text]
  20. Vaisius, A. C., and Weiland, T. (1982) Biochemistry 21,3097-3101 [Medline] [Order article via Infotrieve]
  21. Bhargava, P., and Chatterji, D. (1989) FEBS Lett. 248,195-200 [CrossRef]
  22. Altmann, C. R., Solow-Cordero, D. E., and Chamberlin, M. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,3784-3788 [Abstract]
  23. Johnson, T. L., and Chamberlin, M. J. (1994) Cell 77,217-224 [Medline] [Order article via Infotrieve]
  24. Price, D. H., and Parker, C. S. (1984) Cell 38,423-429 [Medline] [Order article via Infotrieve]
  25. Sluder, A. E., Price, D. H., and Greenleaf, A. L. (1988) J. Biol. Chem. 263,9917-9925 [Abstract/Free Full Text]
  26. Kedinger, C., Gniazdowski, M., Mandel, J. L., Gissinger, F., and Chambon, P. (1970) Biochem. Biophys. Res. Commun. 38,165-171 [Medline] [Order article via Infotrieve]
  27. Lindell, T. J., Weinberg, F., Morris, P. W., Roeder, R. G., and Rutter, W. J. (1970) Science 170,447-449 [Medline] [Order article via Infotrieve]
  28. Jacob, S. T., Sajdel, E. M., and Munro, H. N. (1970) Nature 225,60-62 [Medline] [Order article via Infotrieve]
  29. Johnson, K. A. (1993) Annu. Rev. Biochem. 62,685-713 [CrossRef][Medline] [Order article via Infotrieve]
  30. Rudd, M. D., Izban, M. G., and Luse, D. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,8057-8061 [Abstract]
  31. Feng, G., Lee, D., Wang, D., Chan, C., and Landick, R. (1994) J. Biol. Chem. 269,22282-22294 [Abstract/Free Full Text]
  32. Nudler, E., Goldfarb, A., and Kashlev, M. (1994) Science 265,793-796 [Medline] [Order article via Infotrieve]
  33. Chamberlin, M. J. (1994) Harvey Lect. 88,1-21
  34. Shirai, T., and Go, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,9056-9060 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.