Jan 21, 2016 - in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 8 Trainee .... ATP to ITP) and co-chromatographed with plasmid DNA on.
Vol. 269, No. 3, Issue of January 21, pp. 2299-2306, 1994
Tm Jamwu. OF B m m l c a CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.
Hydrolytic Cleavage of NascentRNA in RNA Polymerase I11 Ternary Transcription Complexes* (Received for publication, August 16, 1993, and in revised form, October 7, 1993)
Simon K. Whitehall$, CarolyneBardelebed, and GeorgeA. Kassavetisn From the Department of Bio1og.y and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0634
Highlypurifiedyeast RNA polymerase I11 ternary cipally the nucleosomes, represent potential obstacles to RNA chain elongation. Indeed,there is some evidence to suggest that complexes were found to possess a hydrolytic chain reRNA from its nucleosomes can provide some impediment to RNA chain elontractingactivitythatcleavesnascent 3“OH end. Most of the shortened transcripts were ca- gation by RNApolymerase (pol)’ I1 (3,4) andmore so for pol I11 pable of resuming RNA chain elongation, indicating that (5,6 ) . Sequence-specific DNA-binding proteins may also intertheyremainstablyassociatedwiththeenzyme-DNA fere with the passageof RNA polymerase along the template. complex. Analysisof the products of cleavage indicatedDNA-bound Escherichia coli lac repressor and EcoRI block that retraction primarily occurred in dinucleotide intranscript elongation by E. coli RNA polymerase and pol I1 crements, but that mononucleotides were also excised at (7-9). In addition, elongating ternary complexes have a high lower frequency. The ribonuclease activity was totally probability of becoming trapped at certain sequences within dependent on the presence ofa divalent cation and wastranscription units such that they can neither elongate nor stimulated by the addition of non-cognate ribonucleodissociate (reviewed in Refs. 10, 11). The elongation factor tides. The inclusion ofATP in the reaction enhanced TFIIS (also called SII; 121, which interacts with the largest both the rate and extent of transcript cleavage. Evidence suggesting that the hydrolytic activity is intrinsic subunit of pol I1(13, 141, greatly mitigates transcriptional stallto RNA polymerase I11 and factor-independent is also ing at such intrinsic arrest sites(15-17). This accessory factor of the obstruction provided by a lac presented. Transcript cleavage by RNA polymerase 111 also facilitates readthrough repressor-operator complex ( 8 ) . Furthermore, TFIIS has been ternary complexes appears to be more closely related to the intrinsic nucleolytic activityof vaccinia virus RNA shown to be a determinant (possibly the sole determinant) of the nucleolytic activity in pol I1 ternary complexes. In theprespolymerase ternary complexes than to TFIIS-dependent cleavage that has been described RNA for polymerase II ence of TFIIS, the polI1 ternary complex cleaves RNA in a processive 3’ + 5‘ manner,releasing mononucleotides, diternary complexes. nucleotides, and to a lesser extent, trinucleotides and even longer products (18-21). Importantly, there is strong evidence Elongation of RNA chains in transcription is catalyzed by to indicate that this TFIIS-stimulated nucleolytic cleavage is RNA polymerase within a highly processive enzyme-RNA-DNA essential for TFIIS-potentiated readthrough past intrinsic or ternary complex. The substratesfor elongation are ribonucleo- protein-induced blocks to RNA chain elongation (2, 22, 8). Hydrolytic cleavage is notsolely a n eukaryotic phenomenon. side triphosphates that are sequentially transferred to the 3”OH end of the nascent RNA through the formation of 3’-5’- Indeed, transcript cleavage was first demonstrated in E. coli phosphodiester linkages and the release of pyrophosphate. The RNA polymerase ternary complexes (23). Two newly identified ternary complex also mediates the reversereaction, pyrophos- elongation factors, GreA and GreB, are responsible for stimuphorolysis, yielding back the substrates of elongation and a lating thiscleavage reaction of E. coli RNA polymerase ternary transcription complexes (24, 25). In the presence of GreA, the shortened RNA chain. It hasrecently been demonstrated thata number of different nascent RNA is cleaved in steps of two or three nucleotides, of longer (up RNA polymerases in ternary complexes have a second RNA whereas theaction of GreB results in the release chain retracting activity (reviewed in Ref. 1).In this process, to nine nucleotides) RNA products (25). Whether nucleolytic retraction of the RNA polymerase along the DNA template RNA cleavage is an intrinsicproperty of RNA polymerase that occurs through hydrolytic cleavage of the growing end of the is stimulated by accessory factors such as GreA, GreB, and nascent transcript, generating short RNA products. It has been TFIIS, or whether the nucleolytic activity resides in these facpostulated that this hydrolytic retraction provides a mecha- tors remains a point of uncertainty (1).Here we report the nism for overcoming elongation arrest and that theprocess in identification and characterizationof a divalent cation-dependwhich the paused complex retracts and subsequently resumes ent 3’ 5‘-exonuclease activity associated withhighly purified elongation facilitates multipleapproaches to the block in tran- Saccharomycescerevisiae RNA polymerase I11 ternary complexes. We find that pol I11 stalled on a SUP4 tRNA% template scription(2). Eukaryotic RNA polymerases must transcribe DNA that is packaged into chromatin,whose components, prin- is capable of cleaving nascent RNA in both mono- and dinucleotide steps.Nucleolytic cleavage is stimulatedby the presence of * This work was supported by Grant GM18386 from the National non-cognate ribonucleoside triphosphates. We also present eviInstitute of General Medical Sciences (to E. P. Geiduschek). Thecosts of dence that a dissociable, TFIIS-like factor is not involved with publication of this article were defrayed in part by the payment of page nucleolytic RNA chain retraction by pol 111. charges. This article must thereforebe hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: pol, RNA polymerase; nt, nucleotideb); t Recipient of a Human Frontier Science Program Fellowship. TLC, thin layer chromatography; bp, base paifis); ATPyS, adenosine 8 Trainee of Grant 07317 in Cell and Molecular Biology from the 5’-O-(thiotriphosphate); AMP-PCP, P,y-methyleneadenosine5“triphosNational Institute of General Medical Sciences. phate; AMP-PNP, P,y-imidoadenosine 5’-triphosphate; AMP-CPP, a$7 To whom correspondence should be addressed. methyleneadenosine 5’-triphosphate.
-
2299
2300
RNA Polymerase Ill-associated Riboexonuclease EXPERIMENTALPROCEDURES
Pst I 3'overhang
normal +1 S t a r t site
1 I+ Materials-Unlabeled fast performance liquid chromatography puriGGTACTCTTTCTTCAACAATTAAATACTCTCGGTAGCCAAGTTG.. . fied ribonucleotides (Pharmacia LKB Biotechnology, Inc.), [CX-~~PICTP ACGTCCATGAGAAAGAAGTTGTTAATTTATGAGAGCC-. .. (New England Nuclear), shrimp alkaline phosphatase (U. S. Biochemibox A b a 1 cal Corp.), dinucleotides, and yeast inorganic pyrophosphatase (Sigma) 00 were purchased. FIG.1.Formation of C31 ternary complexes. The 5"flanking seHighly purified RNA polymerase I11 was prepared as described (26, quence of the SUP4 tRNA* gene (plasmid pLN4031) is shown extend27). The DNA template used in this study was pLN4031, whichcontains ing from the 3"overhang that isgenerated by cleavage with PstI to the the SUP4 tRNA* gene (28,29), cleaved with PstI and HindIII, followed B'-proximal box A promoter element. Transcription was primed with by extraction with phenol-chloroformand precipitation with ethanol. GpG (underlined) to initiate at the single strand-double strand junction Purified %nary Complexes"Ternary complexes stalled at position and elongated with ATP, [CX-~~PICTP, and UTP to position C31. C31 (see Fig. 1)were typically formedin 100-pl reaction mixtures that contained 50-100 fmol of active RNA polymerase 111, 50 fmol of PstI- ing a 31-nt nascent transcript were formed by priming tran~ 25 p , ATP, ~ and HindIII-cleaved pLN4031 DNA template, 400 p , GpG, scriptional initiation with the dinucleotide GpG at a PstI-gen100 p , UTP, ~ and 5 p , ~ [CX-~~PICTP (200,000 countdmidpmol) in trannatural scription buffer (40 m~ Tris-HC1, pH 8 , 6 m MgCI,, 100 m~ NH4CI, 3 erated 5'-end located 14 bp upstream of the m~ dithiothreitol, 160 p g / d bovine serum albumin, 5% glycerol) for 45 transcriptional start site of the SUP4 tRNAmgene and allowmin at 20-21"C. The reaction was terminated by the addition of ing RNA chain elongation to occur with a ribonucleotide mixNa3EDTAto 10 m followed bygel filtration on a 1-ml Sepharose CL2B ture lacking GTP(Fig. 1).Incorporation of c~-~~P-labeled CTP in column equilibrated in transcription buffer without MgCI,. Ternary this elongation mix facilitated uniform labeling of the 31-nt complexes were harvested by collecting 2-dropfractions (-30 pl). The transcript at positions C5, C7, C11, C14, C17, C27, C29, and first threefractions containing radioactive label were pooled,diluted in C31. The products generated by this initiation protocol are column bufferas required, and split into aliquots. Transcript truncation shown in lane 1 of Fig. 2, panel a . Besides the predicted C31 was performed at 20 "C in 15 pl final volume. Reactions were initiated by the addition ofMgCl, to 7 m and terminated by the addition of RNA product2 several smaller RNA products were generated. , by addition of 8 pl of 98%deionized Na3EDTA to 10 m ~ followed Nearly all of these shorter RNA products were abortive (reformamide and heating a t 100 "C for 3 min. Samples were analyzed on leased from the complex) since they were separated from the 20% (19:l or 39:l acrylamidehisacrylamide) polyacrylamide gels conlinearized plasmid DNA, containing the intact ternary comtaining 8 M urea with 50 m~ Tris borate and 1m~ Na,EDTA, as running buffer. The size of the C31 transcript was confirmedby the two-nucleo- plexes, upon addition of EDTA and passage througha Sepharose CL2B column in buffer lacking M$+ (lane 2).These >lO-nt tide extension generated upon addition of GTP to isolated C31 ternary complexes (see Figs. 1and 4). Shortened transcripts generated by Mg2+- long abortive RNA products were unexpected since ternary induced cleavage were identified by counting bands shorter than the complexes containing RNA chains of 10, 12, and 17 nt were C31 initial complex on over-exposed autoradiograms. This indexing was previously found to be highly stable (32, 26, 33, and data not confirmed fortranscripts shortened to nt A19 by observing the effect of shown). Nevertheless, Sepharose CL2B chromatographyalthe presence of ATP and UTP individually on band intensity. ATP enhanced retraction signals at A26,A24, and A19 and UTP enhanced lowed the generation of a nearly homogeneous C31 substrate signals at U28 and U25. When an analysis of the small products of suitable for examination of nucleolytic transcript shortening. A small proportion of complexes with a 32-nt nascentRNA chain transcript cleavage was desired, samples were analyzed on 28% polyacrylamide (25:3 acrylamidehisacrylamide), 7 M urea sequencing gels was also generated (presumably due to trace deamination of with 89 m~ Tris borate, 2 m~ NazEDTA as running buffer (20). Auto- ATP to ITP) and co-chromatographed with plasmid DNA on radiograms were made from wet gels with or without an intensifying Sepharose. A majorityof the C31 ternary complexes remained screen. active through column isolation as evidenced by their ability to Thin Layer Chromatography (TLC) of Reaction Products-Transcript retraction reactions were carried out as described above. Samples were be elongated to full-length, terminating at the natural termianalyzed (30) by directly spotting aliquots (10 p1) onto a polyethyl- nation signal -120 bp downstream upon simultaneous addieneimine-cellulose TLC plate (Sigma), which was developed in either tion of MgC12 and allfour NTPs (lane 3 ) .Addition of MgC12 to 0.5 or 1 M LiCl until the solvent front had migrated approximately 15 7 m~ in theabsence of NTPs resulted in shorteningof the C31 cm. The TLC plates were subjected to autoradiography with an inten- RNA transcript, generating a 29-nt RNA species within 5 s of sifying screen. incubation (lane 4 ) . After a 5-min incubation, 27- and 26-nt Mono- and Dinucleotide Standards-The CMP standard was proRNA were also detectable(lane 8).After 30min of incubation in (6000 Ci/mmol) with 3 pl duced by mixing 1 pl of 0.066 p , ~[cx-~*P]CTP of 1 N HCI and incubating a t 100"Cfor 10 min. The reaction was the presence of M$+, the 27-nt RNA had become the predomiincubated on ice for a further 10 min before the addition of 3 pl of 1 N nant shortenedspecies. Longer exposures (not shown) of these NaOH. autoradiograms also identified some smaller (c20nucleotides) Dinucleotides (UpC, CpU, ApC) were 5"labeled in SO-pl reactions RNA. RNAs shorter than C31were also generated by chasing containing 10 nmol of NpN, 5 units of T4 polynucleotidekinase (U. S. stalled ternarycomplexes to full-length (lane 3 ). This suggests Biochemical Corp.), and 12.5 pmol of [y-32PlATP(6000 Cilrnmol) in 70 m~ Tris-HC1, pH 7.5, 10 m~ MgCl2, and 5 m~ dithiothreitol. The reac- that instead of resuming elongation a subpopulation of C31 complexes retracted in thepresence of M$+ and allfour NTPs. tions were incubated at 37 "C for 30 min, cold ATP was added to 6 p~ followed by incubation at 37 "C for a further30 min. All standards were Moreover, the fact that these transcripts were considerably diluted in column buffer (40m~ Tris-HC1, pH 8, 100 m~ NH4C1, 3 m~ shorter than 27 nt implies that ribonucleotides affected the dithiothreitol) before use. backward reaction (and is examined further below). Neverthe-
'
less, the data suggested the action of a 3' + 5"nuclease analogous to thehydrolytic transcript cleavage previously observed Purified pol I11 is capable of initiating transcriptionsite spe- with E. coli, pol 11, and vaccinia virus RNA polymerases (2, 19 cifically and efficiently at 3'-overhanging DNA ends generated 22, 25, 31). No ribonucleolytic activity was detectable upon by restriction endonuclease cleavage (28). We have used this addition of pol 111, MgC12,and theDNA template to labeled C31 property of pol I11 to examine whether it contains an intrinsic RNA not associated in a ternary complex (data not shown). 3' 4 5'-ribonuclease activity, as previously observed with vacWhen all four NTPs were added after5 and 30 min of M e cinia virus RNA polymerase (31),or whether a separable elon- induced retraction the29-nt RNA containing ternary transcripgation factor like TFIIS for pol I1 or GreA and GreB for E. coli tion complex was competent to resume RNA chain elongation, RNA polymerase is required for efficient RNA chain retraction (2, 22, 25). By convention, stalled complexes are identified according to the Ternary (pol 1II.DNA.RNA)transcription complexes contain- nucleotide at the 3' end and the length of the transcript. RESULTS
RNA 111-associated Polymerase Riboexonuclease a
M e
- - - - . - - - - - + + + + + + + + + + - + - - - - - + - + - - + + + + + + + + - - - 5" 1 5 1' 2.5'5' 5' 30' 30'
PreCL2B + PostCL2B ChaseMg2"
Time
2301 PPI PPI-ase
- + - - + + + " + " + +
--
- + + - +
-Full
Length
-C31
-
--
c29 " c 2 7
1 2 3 4 5 6 7 -c31
" " " I . ">-
- C29 ;C27 A26
FIG.3. Influence of pyrophosphate on transcript cleavage.Purified C31 ternary complexes were incubated for 5 min after thefollowing additions: lane 1, no addition; lane 2, 7 m~ MgCl2; lane 3, 1 mM pyrophosphate; lane 4, 2.85 units of pyrophosphatase; lane 5, 7 m~ MgClz and 2.85 units of pyrophosphatase; lane 6 . 7 m~ MgClz and 1m~ pyrophosphate; lane 7, 7 m~ MgClZ, 1 m~ pyrophosphate, and 2.85 units of pyrophosphatase.
1
3 4 5 6 7 8 9 1 0 1 1
2
b A,C,U G,A,C,U
Mg2+
-
+
- -
+
+ + + "full length
1
2
3
III ternary complexes. Panel a , arrested C31 ternary complexes were formed (lane 1 ) and purifiedby Sepharose CL2B chromatography (lane 2 ) as described under "Experimental Procedures."C31 complexes were elongated with 0.1 m~ NTPs and 7 m~ MgClz for 2 min (lane3 ) . Transcript cleavage (retraction) was initiated with 7 m~ MgClZfollowed by incubation for the times indicated (lanes 4-11 ). After incubation with MgCl, for the indicated times, complexes were allowed to resume chain elongation by providing 0.1 m~ NTPs for 2 min (lanes 9 and I 1 ).Panel b, retraction of C31 complexes was performed for 5 min a s described above (lanes 13). Reactions were chasedfor 2 min by adding ATP, CTP, and UTP (lane 2 ) or all four NTPs (lane 3 ) to 0.1 m~ final. FIG.2. ngneCript cleavage by purified pol
generating full-length product (lanes 9 and 11, respectively). The residual amount of the C31complex that remained after 30 min of incubation in the presence of M$+ (lane 1 0 ) was very similar to the amount of apparently inactive C31 complexes complex isolation (lane that were unable to chase after ternary 31, and also had not chased after 30 min of incubation with M e (lane 11 ). In a separate experimentaddition of ATP, CTP,
and UTP after a short period of Me-dependent chain retraction extended the 29-nt RNA back to 31 n t (panel b, compare lanes 1 and 2 ) , confirming that the Me-induced shortening had occurred at the 3'-end of the nascent chain. A significant fraction of C27 and A26 RNAgenerated by M e treatment was incapable of resuming elongation after 5 and 30 min of M e treatment, and the proportion of these unchaseable transcripts increased withtime (panel a, lanes 9 and 11 ).The failureof the C27 and A26 RNA to restart elongation resulted from disruption of the complex upon Me-induced chain retraction to these positions. Rechromatography of complexes on CL2Bafter a 5-min incubation inthe presence of M e showed that whereas the 31- and 29-nt RNA chains co-eluted with plasmid DNA in the excluded volume, the 27- and 26-nt long RNA chains hadevidently released since they eluted in the included volume (data not shown). In experiments not shown, transcript shorteningby the pol 111ternary complex was observed over a broad range ( 1-50 mM) of M e concentrations. Furthermore, other divalent cations could substitute for M e in supporting thiscleavage reaction: retraction in the presence of 7 mM Mn2+resulted in a similar distribution of shortened RNAs to that observed with M e after 5 min of incubation (that is to C29 and C27 in comparable amounts), while Zn2+ or Co2+ addition to 7 mM yielded transcripts predominantly shortened by two nucleotides but not significantly more. A U30 transcript was also observed upon Co2+addition. Littleor no transcript truncation was induced by Ca2+. Although transcript cleavage occurred in theabsence of exogenously added pyrophosphate, i t remained conceivable that RNA chain retraction was pyrophosphorolytic due to low levels of endogenous pyrophosphate bound to, and co-purifying with, DNA-bound polymerase. This does not appear to be the case, however, as RNA shortening observed upon addition of Mg2' to isolated C31 complexes (Fig. 3, lane 2) was not increased by 1 mM pyrophosphate (lane 6).A time course of RNAchain retraction alsoshowed no significant increasein therate of retraction upon inclusion of pyrophosphate (data not shown). In addition, RNA shortening in the presence of M e was not inhibitedby a large excess of inorganic pyrophosphatase(lane 5).Thus, these results strongly indicatethat theretraction process occurred by a mechanism other thanpyrophosphorolysis. The product analysis shown below confirms this assessment. Divalent metal ion-induced transcript cleavage by vaccinia virus ternary complexes has been demonstrated to be stimulated by CTP (31). We therefore determined the influence of
RNA Polymerase 111-associated Riboexonuclease
2302
a
Chase
-
+
Mg2'
-
-
NTP
- - -
-
- -
+
+
+
-
+
+
"-
- - -
-
-
Mg" Mg"+ ATP ATP
-
Mg2++ + PPI
ATP UTP CTP GTP ATP UTP CTP GTP
-- C27 -A24
C29
-A26
Time
0.252.5
5 0.252.5 5 0.252.5
5
(mln)
FIG.5. Influence ofATP and pyrophosphate on the kinetics of transcript cleavage. Retraction was performedby incubating C31 ternary complexes (unmarked lane) with 7 rn MgClz for the times indicated, either alone, with ATP (1 rn), or with ATP (1 m ~ ) and , pyrophosphate (1mM).
low levels of W,due to deamination of CTP, also would generate this result if retraction occurred in dinucleotide increments followed by reincorporation of both CMPand UMP. GTP 1 2 3 4 5 6 7 S 9 1 0 1 1 addition allowed the stalled ternary complex to extend the RNA chain to position G33 as predicted by the sequence (lane 7). Addition of each nucleotide in the absence of Mg2' had no b A,C,U + detectable effect on chain retraction (lanes 8-11 1. In contrast to the disruption of ternary complexes that oc+ G,A,C,U curred upon Mg2"induced retraction to C27 and A26, comMg2+ + + + plexes that retracted to A24 (and C29) upon addition of M e and ATP (Fig. 4b,lane 1 ) remained predominantly intact; adATP + + + dition of ATP, CTP, and UTP extended the transcript back to C31 (lane2 ) and addition of all four NTPs generated full-length full length product (lune 3). ATP-enhanced retraction, nevertheless, resulted in mostly inactive RNA a t positions U28, C27, and A26 (compare lanes 2 and 3 with 1). If the presence of (the cognate substrate) ATP stabilized complexes retracted to A24 (against reiterative retraction occurring in increments of one to three nucleotides to U21, A22,andA23), it clearly was unable to do so at position A26 (where ATP is the only cognate nucleotide when retraction occurs in mononucleotide increments to U25). Thus, when retraction oftwo or seven nt occurred, the transcript remained associated with pol 111, whereas retraction of three, four, or five nucleotides led to the formation of inactive complexes. Evidently it is not simply the case that pol I11 releases the transcript after retracingits path along DNA bymore than 1 2 3 two nucleotides. Indeed, polI11 may undergo conformational FIG. 4. Effect of ribonucleotides on retraction by pol I II.Panel a , C31 ternary complexes were column isolated (lane 1 ), to which indi- changes during retraction that results in the formation of eividual nucleotides (indicated above the lane) at 1 m~ final concentra- ther stable or metastable ternary complexes depending upon tion were added either with (lanes 4-7) or without (lane 8-11) 7 m~ its position on the template. MgC12, and incubated for 5 min or immediately chasedby the addition Investigation of the kinetics of retraction showed that ATP of 0.1 m~ NTP and 7 m~ MgClz (lane 2). Panel b, ATP-enhanced retraction of C31 ternary complexeswas performed by adding 1 m~ ATP increased the rateof RNAcleavage, both in regard to the extent and 7 m~ MgClz and incubatingfor 5 min (lanes 1 3 ). Transcript cleav- of truncation and theproportion of the C31 complex converted age reactions were subsequentlytested for retention of RNA chain elon- to shortened RNA (Fig. 5; compare 0.25 min 2 ATP). Surprisgation activity by adding ATP, UTP, and CTP (lane 2) or all four NTPs ingly, the addition of pyrophosphate to 1 m~ substantially re(lane 3)and incubatingfor 2 min. All chase nucleotides wereat 0.1 m~ final concentration, exceptfor ATP which was approximately1 m ~as, versed the stimulatory influence of ATP, such that the rateof transcript shortening was reduced and the distribution of rea result of its inclusion in the transcript cleavage reaction. tracted transcripts reverted toward that observed in the presnucleotides on RNAcleavage within the pol I11ternary complex ence of Mg2' alone. This pyrophosphate-generated effect was (Fig. 4).In these experiments nucleotides were added to a final not observedwhen inorganic pyrophosphatase was also inconcentration of 1m ~The . presence ofATPhad a marked effect cluded in thereaction mixture, supporting the contention that upon transcript truncation, increasing the extent of retraction, the phenomenon was due to pyrophosphate (data not shown). with A26 and A24 RNA becoming the predominant species While the molecular basis for pyrophosphate-mediated inhibi(panel a, lane 4). W also influenced the extent of retraction tion of ATP-enhanced chain retraction is not clear,it resembles and the distribution of transcripts, generating mainly RNA the effect of pyrophosphate on transcript truncation by the species U28, A26,and U25 (lane 5).By comparison, CTP addi- combined action of pol I1 and TFIIS (19). tion decreased the appearance of shorter transcripts, and RNA In order to assess whether ATP hydrolysis was involved in shorter than C27 was not detectable (lane 6). This result is ATP-enhanced retraction, dATP, ATPyS, AMP-PCP, AMP-PNP, consistent with retraction occurring in mononucleotide incre- AMP-CPP, ADP, and A" were substituted for ATP at 1 mM ments in which removal of the 3'-CMP residue would be ac- final concentration (data not shown). ATPyS substituted eficompanied by reincorporation of CMP. However,the presence of ciently for ATP; all other nucleotides except AMP and dATP
-
-
-
RNA Polymerase 111-associated Riboexonuclease Chase Mg2*
-
-
+
-
+ “full
m
length
-c31 -C29 “c27
1
2
3
FIG.6.Sarkosyl treatment of ternary complexes. Ternary com-
plexes were formed and purified as described under “Experimental Procedures” exceptthat Sarkosyl was addedto 0.3% prior to Sepharose CLBB chromatography. The “Sarkosyl-rinsed” C31 complexes (lane 1) were elongated with 0.1m NTPs (lane2 ) or retracted with 7 m MgClz for 15 s (lane 3).
generated weak enhancement of retraction. This weakly enhanced retraction by non-hydrolyzable nucleotides maynot necessarily signify that ATP hydrolysis was not requiredsince the same experiments showed that ADP, AMP-PCP, AMP-PNP, and AMP-CPP were all contaminated with ITP or GTP (as evidenced by trace extension of the original C31 complex to a larger size). A 1% contamination of ATP would have approximated the enhanced level of retraction observed. The requirement of ATP hydrolysis in enhanced retraction therefore does not appear likely, but it is not excluded at this time. RNA cleavage by pol I1 and by E. coli RNA polymerase ternary complexes has been demonstrated to be greatly stimulated by dissociable elongationfactors (reviewed in Ref. 1). Although the preparationof pol I11 used for these experiments was highly purified (27), we could not rule out the possibility that a dissociable factor was responsible for, or stimulated, transcript cleavage. We therefore formed ternary complexes as previously described but addedSarkosyl to 0.3% prior to loading on the Sepharose CL2B column. A similar method has been employed by others to remove elongation factorTFIIS from pol I1 ternary complexes (22). A major fraction of the ternarycomplexes isolated by this procedure (Fig. 6, lane 1 ) remained active and were capable of elongating to full-length (lane 2 ) . Upon presentation with M e , transcript cleavage was detectable after 15 s (lane 3) and was more extensive than that observed for complexes that hadnot undergone Sarkosyltreatment (Fig. 2, lane 5).A similar experiment in which Sarkosyl was added to 0.3% after column isolation of C31 ternarycomplexes for 2 min resulted inconsiderable inactivation of these complexes such that only a small fraction were competent to resume elongation to full-length. We did note, however, that in the presence of 0.3% Sarkosyl equivalent proportions of complexes were capable of retraction and elongation (data not shown). a-Amanitin, a n inhibitor of RNA chain elongation, has been shown to inhibit RNA chain retraction of RNA polymerase I1 (34,22,19). Although a-amanitin is not aneffective inhibitor of RNA chain elongation for yeast pol 111, tagetitoxin is (35).Addition of tagetitoxin to 8000 unitdml abolished production of full-length transcripts upon addition of nucleotides to columnisolated C31 complexes, but did not inhibit Mg2“induced retraction (data not shown). We did note that tagetitoxin appeared to changetheregister of retraction at this high concentration, such thatMg2”induced bands corresponding to
2303
U30 and U28 wereobserved in place of C29 and C27. However, product analysis,similar to that described below, demonstrated that addition of tagetitoxin resulted in themisincorporation of a pyrimidine nucleotide prior to Me-induced retraction. Likewise, addition of calf intestinal alkalinephosphatase to a reaction mixture containing tagetitoxin, prior to the addition of MgC12, but not after, eliminatedthe tagetitoxin effect (presumably due to the hydrolysis of the contaminatingNTP) (data not shown). We used polyethyleneimine thin layer chromatography (30) to characterize the products of the retractionprocess. Isolated C31 complexes uniformly labeled with CTP were exposed to M e to induce transcript truncation, and a portion of the reaction mixture was spotted onto a TLC plate that was develto find labeled CTP as the oped in 1M LiCl (Fig. 7a). The failure reaction product argues againstcleavage via pyrophosphorolysis (lane 4 ) . Products co-migrating with CMP and dinucleotide markers were observed instead. We conclude that transcript cleavage in the pol I11 ternary complex occurs via hydrolytic cleavage rather than by pyrophosphorolysis. The use of 0.5 M LiCl as the solvent allowed greater resolution of mononucleosidemonophosphates from dinucleoside diphosphates, predicted to be the hydrolysis products generated by incremental cleavage by one or two nt from the 3’-end of the RNA (note that pUpC and pCpU migrate identically in this system; see sequence in Fig. 1).Analysis of the reaction products in thisway revealed that transcript truncation mostly yielded dinucleotides and, to a lesser extent, CMP (Fig. 7b, lane 4; 3’-UMP should migrate faster). Although inorganic phosphate also migrates between CTP and CMP in thissystem, other data indicate it is not a major hydrolysis product (see below). The inclusion of ATP in the reaction that was shown above to enhance cleavage, led to a decrease in the proportional yield of CMP (lane 5). We conclude that RNA chain retraction bypolI11 ternary complexes can proceed in single nucleotide and in double nucleotide steps. We sought to characterize the dinucleotide products further using high resolution polyacrylamide gel electrophoretic analysis. It is possible to resolve different species of dinucleotides on 28% polyacrylamide gels, the hierarchy of mobility being YpY > YpRlRpY > RpR (20). Thus, while it is possible to distinguish between dinucleotides with differentbase compositions it isnot possible to resolve them by sequence (20). Portions of the reaction mixtures described in Fig. 7, a and b, were examinedusing this method, and the results are shown in Fig. 7c. Consistent with the polyethyleneimine-TLC analysis, the major products of retraction induced by M e were found to be CMP and a dinucleotide that co-migrates with pUpC/pCpU (lane 4 ) . As also observed by TLC analysis, ATP reduced the formation of CMP product. A putative dinucleotide species with a slightly lower mobility than pUpC/pCpU was also detectable, but this was presentat a much lower level than themajor dinucleotide product. Based upon its mobility we do not believe that this represents a trinucleotide. It is possible that thisproduct is the result of pol I11 retraction pastC27, which is seen at a low level in these experiments, or less probably from low level incorporation of IMP at position 32 in the startingreaction with subsequent generation of pCpI. We also investigated the kinetics of product formation, demonstrating that the pUpC/pCpU dinucleotide is themajor product 15 s after the initiation of transcript truncation(Fig. 8, lane 3).A small amountof CMP was also observed at this time. The other slowly migrating dinucleotide product formed more slowly, becoming detectable after 2.5 min of RNA chain retraction. If this slowly migrating product is pCpI, its rate of excision as the first product to be formed must be slow. Removal of 5”phosphate from these products with alkaline phosphatase
RNA Polymerase 111-associated Riboexonuclease
2304
a
C31 Complex I
Mg2' Mg2* +ATP
-
CMP CTP
I
PCPU
-
CTP
- upc
- origin 1
2
4
3
b
5
6
7
C31 Complex I
-
CMP CTP
Mg2. I +ATP PCpU
Mg2'
PAPC -pupc/pcpu CMP -
- ATP
- CTP 1
1 4 2
1
C
3
6
5
-
7
C31 Complex CTP
CMP
-
ATP Mg Mg UC
AC CU
- PAPC
- pcpu I pupc
- ATP
CMPCTP1
2
3
4
5
6
7
8
RG.7. Products of transcript shortening. Panel a , Sepharosepurified C31 ternarycomplexes were retracted with 7 nw MgCl, with or without 1 nw ATP for 5 min. A portion of each reaction mixture was spotted onto a TLC plate which was developed in 1 M LiCl. CTP, CMP, pCpU, and pApC were chromatographed as markers. Lane 1, CTP; lane 2, CMPlane 3, C31 ternary complexes; lane 4, C31 ternarycomplexes
2
3
4
5
6
7
8
9
10
1112
FIG.8. Time course of product formation. Isolated C31 ternary complexes were retracted with 7 m MgCl, for the times indicated above the lanes (lanes 2 4 , 11, and 12). Markers were included as standards as indicated (lanes 1and 7-10; the dinucleotides CU, AC, and UC were 5'-phosphorylated). Transcript cleavage reactions were also treated with 10units of shrimp alkalinephosphatase for 2 min a t 20 "C (lanes 11 and 12). The sample for lane 12 contains ternary complexes, which were inactivated by heating at 100 "C for 2 min prior to the addition of 7 nw MgClz and 10 units of shrimp alkaline phosphatase. was used to distinguish between dinucleotides with external and internal label. Since these transcripts were labeled with [(Y-~~PICTP, pUpC but not pCpU should have label that is phosphatase-resistant. Removal of the 5"phosphate from dinucleotide diphosphates has been previously demonstrated to result in a large decrease in mobility of the dinucleotide in this gel system (20, 21). Upon treatment with alkaline phosphatase, most of the dinucleotide-incorporatedlabel remained, but was contained in a much slower migrating compound (lane 11 ). When C31 complexeswere boiled prior to the addition of MgClz and alkaline phosphatase no RNA cleavage products were generated (compare lane 12 with lane 2 ) indicating that the new product generated in lane 11 was not the result of an endonuclease contaminant in the alkaline phosphatase. These results are consistent with the conclusion that most of the dinucleotide product of 3' +.5' RNA chain retraction contains an internal label and is therefore pUpC. Consistent with this conclusion, when transcripts were labeled with [cY-~~P]UTP most of the label in the dinucleotide retraction product was lost upon treatment with phosphatase (data not shown). This result is also consistent with the assumption that theobserved dinucleotide cleavage products are 5'- rather than 3'-phosphorylated,
+ MgCl,; lane 5, C31 ternary complexes + MgCI, + ATP, lane 6, pCpU, lane 7, pApC. Panel b, as for panel a , except that the TLC plate was developed in 0.5 M LiCl. Panel c, analysis of products on a 28%sequencing gel. Reactions were as described for panels a and b. Lane 1, CTP; lane 2, CMPlane 3, C31 ternary complexes; lane 4, C31 ternary complexes + 7 m MgC1,; lane 5,C31 ternary complexes + 7 m MgC1, + 1 m ATP; lane 6 , pUpC; lane 7, pApC; lane 8,pCpU.
RNA Ill-associated Polymerase
Riboexonuclease
2305
through bound TFIIIC (a protein equivalent in size to pol III), and the intrinsic nucleolytic activity present in highly purified pol 111 ternary complexes is consistent with the rapidity with which purified pol I11 surmounts the TFIIIC-DNA complex obstacle. The rate of retraction observed in this study, in which slightly more than half the active ternary complexes have retracted two nt by 5 s (Fig. 21, is apparently incompatible with chain retraction playing a significant role in limiting the DISCUSSION TFIIIC-dependent delay of elongation to 0.2 s. However, the The potential to cleave nascent RNA by a hydrolytic mecha- presence of NTPs clearly stimulates the rateof retraction by pol nism has recently emerged as a property of several prokaryotic I11 (Figs. 4a and 51, and one might expect that the rate of and eukaryotic RNA polymerase ternary complexes. The iden- retraction also has a sequence positional parameter. The aptification of such an activity associated with polI11 ternary pearance of aborted,
