J. Mol. Biol. (1992) 226, 47-58

Formation

of Open and Elongating Transcription by RNA Polymerase III

Complexes

George A. Kassavetist, Jaime A. Blanco, Terence E. Johnson and E. Peter Geiduschek Department

of Biology University

9500 Gilman

and Center for Molecular San Diego La Jolla, CA 92093-0634,

Genetics

of California,

Drive,

(Received 2 October 1991; accepted 10 February

U.S.A. 1992)

The Saccharomyces cerevisiae transcription factors (TF) IIIB and IIIC assemble onto their respective DNA-binding sites on the SUP4 tRNATyT gene at 0°C. RXA polymerase III specifically associates at 0°C with this TFIIIC-TFITIB-DNA complex to form a stable ‘.closed” promot’er complex in which the D?iA surrounding the transcriptional start retains its duplex form. Promoter “opening” is a temperature-dependent and readily reversible process that involves up t,o 22 unwound base-pairs of DNA, and can be followed by analyzing the hyperreactivity of thymine to KMnO, oxidation. This promoter opening increases progressively from 10°C to 4O”C, with at least two regions within the transcription bubble appearing to melt’ independently. In contrast, the temperature dependence of forming an initiated transcription complex containing a 17 nucleotide nascent RNA chain displays a sharp transition between 10°C and 15°C. When RNA polymerase initiates transcription under conditions that limit the nascent RNA chain to less than six nucleotides. there is no displacement of the transcription bubble. These transcription complexes are distinguishable from “open” promoter complexes in their maintenance of the transcription bubble at O”C, and from transcription complexes with more extended (17 nucleotide) RNA chains in their sensitivity to disruption by heparin. In light of recent results by others that demonstrate a requirement for an RNA transcription factor in a Bombyx mori-based in vitro RNA polymerase III transcription system, we have searched for a comparable component in the 8. cerevisiae-derived system. We show that if an RSA component is required in the yeast-derived system, it is not susceptible to inactivation by massive amounts of micrococcal nuclease, RXase A, or RNase T,.

KeywordA: RNA

polymerase

III; open complexes; transcription factors

1. Introduction

initiation;

footprinting;

genes, and TFIIIA binds to the box C element of 5 S rRNA genes, allowing TFIIIC to follow (for reviews, see Gabrielsen & Sentenac, 1991; Geiduschek & Kassavet’is, 1992). These protein-DXA complexes function, in Saccharomyces cerevisiae, to position a third factor, TFIIIB, directly upst)ream of the start site of transcription. TFIIIB alone. bound to upstream DNA sequence, correctly positions pol III at the transcriptional start for repetitive rounds of transcription (Kassavetis et al., 1990). Much of the research on transcription by pol III has centered on the assembly and properties of these transcription factor-DNA complexes. with less emphasis on the subsequent steps of transcription. Given the great stability of the TFIIIB-DNA complex, and the lack of discernible DNA sequence

RNA polymerase III (pal 111)s transcribes genes coding for a variety of small RNAs: tRNA and 5 S rRNA are its quantitatively predominant products. The major promoter sequence elements of tRNA and 5 S rRPliA genes, which lie within transcribed sequence, are DNA-binding sites of pol III transcription factors (TFIII): TFIIIC binds to the box il and box L3 intragenic promoter elements of tRNA t Author to whom reprint requests should be addressed. $ Abbreviations used: pol III, RXA polymerase III; TF. transcription factor; bp, base-pair(s); nt, nucleotide(s); DTT. dithiothreitol; MNase, micrococcal nurlease; RSA. bovine serum albumin.

47

0

1992 Academic

Press Limited

48

C. A. Kassavetis

specificity in its binding, the rat’e at which a part,icular tRNA gene is t’ranscribed might be determined less by the overall rate of assembling TFTIIB onto upstream DNA sequence than by the subsequent steps of the transcription cycle: (1) binding of RNA polymerase t>o the TFIIIB-promoter complex: (2) localized DNA melting at the transcriptional start; (3) initiation; (4) RSA chain elongation: (5) termination and release of RNA polymerase for another round. Each of these steps has a precise equivalent in prokaryotic transcription: in Escherichia coli. each step can constitut)e a major control point for the regulation of gene expression (for reviews. see McClure, 1985: Yager bl: von Hippel. 1987; Hut, 1987: Gill et al.. 1990). The work present)ed here represents our initial characterization of the steps leading to productive R?r‘A chain elongation by pol TIT. We use several footprinting methods to show that pol III forms a stable promoter complex, and t’hat it effects a localized DNA melting at the transcriptional start to form an open complex. Open complex formation is a tempersture-dependent and readily reversible process. involving up to 22 bp of DNA in the transcription bubble. We also show that pol III that has init)iated transcription but contains RNA chain less than 6 nt long. and pol TIT in a productive ternary complex with a 17 nt nascent transcript have distinguishable properties. A diffusible RNA component has been found to be essential for pol III transcription in a,n in vitro system derived from Bombyx mori (Young et nl.. 1991). \Ve use a footprinting method to show- that. if transcription by pol TII in S. cerevisiue requires such a component, it, is not susceptible to inact’ivation b? massive quantities of micrococcal nuclease (MNase), RXase A or RNase T,.

2. Materials and Methods Plasmids pT%l and pTZ2 containing the yeast SI’P1 tRh’4Ty’ I G62 + C promot)er-up mutant gene. DriA purification and the preparation of I)SA probes. 3’ or 5’ endlabeled at the EcoRT or -YhaT site. of these plasmids have been described (Kasaavetis rt ~1.. 1989. 1990). Dr\‘A-affinity-purified TFIIT(‘. (“bI a(‘ron blue-Sepharosrpurified TFIIIB and Mono Q-purified pal III were prepared as described (Kassavetis et al., 1990). TFIII(: and TFIIIB are specified in fmol of DNA binding activit) (Kassavetis et al.. 1991): pal IT1 is specified in terms of fmol of active molecules for specific transcription and represent,s a minimum estimate (Kassavetis et ~1.. 1989). Ribonucleases A and T, were from BMB: micrococral nurlease was from Pharmacia,-LKB: DBase I (RiYasefree) and Klenow DNA polymerase I were from GIBCO-BRL. Neocuproinr. 1.IO-phenant,hrolinr. and 3-mercaptoproprionic acid were from Aldrich Chemicals. I’ltrapure rNTPs were from BMB or Pharmacia-LKH: [Cc-32P]r,hTPs and dETPs were from NEN DuPont. (b)

Protein

dilutions

TFIIIC. pol III. RNase i4 (after heat inactivation of DNase) and RNase T, were diluted in BIC;A diluent

et al,

(20 mwSa Hepf33 0.2 mM-xa3twrA. (pH 7.X). 7 m&r-MgCl,. 100 m31-XaCl. 10 rn.M-2-rnrr~aptoethanol. 20 “; (v/r) glycerol 1 0.005 O. (v/v) Twrrn-20 (Pierce). 200 pg bovine serum albumin/ml, 1 pg Irupept~in~ml. 1 p’p pepstatin/ml, 05 mM-phenvlmethylsulfonyl fluoride. For the experiment shown in Fig. 9(b). R,lu’asc ‘I’, was pelleted from it,s ammonium sulfate suspension and dissolvttid in BSA diluent, larking Na(‘l. Mic~roc~oc~c~al nuclease was diluted in MXase diluent (the same nlrdium caontaining 20 mzCa(‘12), DBase I w.as diluted RS dr~~ribrtl (Kassavebis rt crl.. 1989).

Incubations lvith transcription proteins and I)NA or nurlease were performed in a total volume of IX or 20 ~1 of transcription buffer containing 10 mz+Tris. HC’I (pH X.0). i mu-Mg(‘l,. 3 m31-DTT. X0 mrv-NaCl. O..i”,, (M.!v) polyp vinylalcohol. 100 pg bovine serum albumin:ml. .i to loo,, (vi\-) glyrrol. Multiple-round transcription was carried out with 100 ng of pT%l D?iA (50 fmol) as t,emplate and was initiated by adding a 5 x NTP solution in transcril)tion hfkr providing %OO/~M-.\TP. 100 ~w-(‘TP. and 2.5~M-[~-~~P]GTI’ (10 cats;min per fmol) 100 ~M-~TP DNase I protecation. (OP),(‘u’ protection. and KiMnO,-oxidation reactions contained 100 ng of p(:ENl (Promega) and 2 to 4 fmol of end-labeled SUP4 t RSA gene probe. (OP),t’u’ protect,ion experiments lacked I)TT in the incubation mixture. Ternary trarwriptiofl complexes were formed by adding an 1I x ?;TP solution providing 200 p&r-ATP and 100 PM-C’TP and VTP. where applicable. Since significant c~ross-rontaminat,ion in these ultrapure nuctleotide stocks (in particular. the deaminat,ion protluc~ts of .4TP and (‘TP, TTP and I’TP. rvspcv~ tively) would lead to nascent RP;A chains that are longrr t’han predicted for the ternary eomplexrs analyzed in Figs 6 and 7. the folloning assags were performed to address the raxt,ent of c.ross-contamination. Electrophoretic~ anal>sis of the 1i nt nascent RSX chain formed upon addition of ATE’, (‘TP and I’TP showed negligible contamination of ATP by TTP or (:TP (data not shown: see for example Fig. 1 of Kassavetis et ~1.. 1989). Contamination of AITF’ and (‘TP by I:TP was assessed on a ditY+rent tt~rnI)late whose transcript8ion initiat~tvl in thr ahsrnce of \-TI’ grnerates a IOnt nascent ItP;.A chain that (‘an tw analyzrd f~l~~c~trophorclti(~all~-.Only a small frac,lion of’ ternary ~~omplrxrs ( dewribed (Kassavcktis et ~1.. 1990). The ~mibined signal 01’ unwound T residues within the open c>omplrx (lid not inc.rease above 13 m#-KMnO, at SOY (data not qhtrwn). Xo quantitative difference brtucrn KJlnO, oxidation of unwound T residues at 30”(’ and 0°C’ was detectable (tlata not shown: see Fig. 6). (OP),(‘u’ cleavage was initiated byadding 2 ~1 of 60 &mercaptopropionic acid and 2 ~1 (ii I mnr-l IO-phenant,hroline. 1 rnM-CuSO, in rapid SUCWSsion to the 20.~1 incubation mixuturr and terminated 30 5 later by adding 2 ~1 of 56 mM-neoc*uproine. Samples wcr was little or no pol ITT-dependent T-oxidation bj KMnO, at 0°C’ (Fig. 3(a)). Above 5°C’. T-oxidation around the transcriptional start (the locaation of the T residues from -9 to + 11 on the non-tralrsc.rit,ed strand is shown at the right of the Figure) increased progressively over a wide temperature range (Fig. 3(a), (b) and (c)). Figure 3(d) shows that the pal TIT-induced strand opening was readily reversed upon

shifting

the

temperature

bacsk

to

O”(‘.

pal

III-DNA complexes formed at 25°C (lane b) lost the enhanced T-reactivity at the t,ranscriptional st’art following a one mmute incubation at 0°C:

RNA Polymerase III

Transcription

51

Complexes

c

0.4 0.2

I

llv

I!

0.0~~““““““““““““““‘~ 2 4 6 8 IO 12 14 16 18 19 20 2224

262830

Figure 4. Rate of open complex formation at 15°C. Plasmid pTZ1. 4 fmol 3’-end-labeled on the non-transcribed strand. was incubated with TFIIIC (17 fmol) and TFTTTB (15 fmol) for 40 min at 21 “C and then incubated at 15°C’ for 10 min. pol III (3 fmol) was added and the incubation continued for the specified times prior to a 30 s KMnO, treatment at 15°C (m). An identical set of reactions was performed where all incubations were at 25°C (0). T-reactivity at the transcriptional start was quantified by densitometry of the autoradiogram. The “zero min” control contains no protein. The 2 time-courses were performed and analyzed as separate experiments. According to Fig. 3(b). the ratio of integrated T residuereactivity in the transcription bubble at 15°C to that at 25°C’ is 1 : 1.X.

(lane c). (The experiment shows that the barrier to pal III-induced strand opening at 0°C is thermodynamic rather than kinetic.) We noted that’ the pattern of KMnO, reactivity was similar but not identical at all temperatures (Fig. 3(c)). Reactivity to KMnO, appeared at T -9 to T -5 at, a slightly lower temperature than at’ T -3 t’o T + I I, but both thermal transitions were quite broad. Evidently, the temperature dependence of KMnO, oxidation of T residues at the t8ranscriptional start (Fig. 3(b)) represents the t’hermal shift of two equilibria, each involving two states of the pol III-DNA complex: a closed (duplex) state that is relatively stable at low temperatures and an open (partly melted) state that is relatively stable at the highest temperatures. We comment further on this issue in Discussion. Figure 4 examines the overall rate of DNA binding and open complex formation at two intermediate temperatures. At both 15” and 25”C, pol III-induced T-reactivity reached a plateau within 20 minutes. confirming that the complexes at or above 15°C had been brought to, or close to. equilibrium.

qf initiated

transcription

IO Reaction

BIndIng time (min)

(a) 4 nalysis

5

complexes

When pol 111 is allowed to initiate transcription on the SUP4 tRNA gene by adding ATP, CTP and UTP. RNA chain elongation halts 17 nt downstream of the start site, before the first G (Kassavetis et al.: 1989; sequence in Fig. 6(b)). The advance of RX-4 polymerase along the template is accompanied by a shift in the location of KMnO,

15

I 20

temperature

I 25

I 30

: 5

(“C)

Figure 5. Effect of temperature on ternary complex formation. Plasmid pTZ1, 4 fmol 3’-end-labeled on the transcribed strand was incubated with TFIIIC (14 fmol) and TFIIIB (8.9 fmol) for 40 min at 21 “C. ATP, CTP and CTP were added to the TFIIIB~TFIIIC-DNA complexes, t)ransferred to the temperatures indicated for 10 min. pol III was then added, and after an additional 20.5 min. a 30 s treatment with KMnO, was performed. Hyperreactivity at T +l to T +lO was quantified as described in Mat,erials and Methods.

oxidation-sensit’ive T residues on both DX A strands (Kassavetis et al., 1990; and see Fig. 6). In the experiment that is shown next, we examined the temperature dependence of ternary complex formation. (We use the terms binary to denote complexes containing only DNA and protein, and t’ernary for complexes containing DNA, protein and RNA). For this experiment, TFIII(C + B)-DNA complexes were preformed at 21 “C and thermally equilibrated at the temperature of the subsequent reaction. RNA polymerase was allowed to bind and elongate RNA chains to nucleotide 17 for 20 minut!es before probing the non-transcribed strand with KMnO,. Quantification of the resulting signals. as described in Materials and Methods, yielded the results shown a sharp transition in in Figure 5, in which T-reactivity was observed between 10°C and 15°C. In a previous study (Kassavetis et al., 1990), formation of a t’ernary complex halted 17 nt downstream from the start site shifted the T residues hyperreactive t’o K&O, on the non-transcribed - 7/+ 7 in the binary strand from base-pair complex to +6/+ 16 in the ternary complex. This is confirmed in Figure 6(a), except that T --9 on the non-transcribed strand of the binary complex at 30°C is also reactive (lane g). When the pol III-containing binary complex is provided with ATP only or with ATP and CTP. nascent RNA chains of lengths two and five are capable of being formed, yet the pattern of T reactivity barely changes from t’hat of the binary complex (lanes h and i. respectively) except that T + 11 becomes slightly more reactive. Because the quantities of active transcription complexes that are formed in these reactions are typically low (of the order of 1 to 10 fmol), it has not been feasible to examine the synthesis of pppApA or pppApApCpApA directly. It therefore remains to be determined whether pol TTT. like E. coli RNA polymerase and pol TT, has an

52

G. A. Kassavetis et al. a

B,C,pIU: No. NTP:

bcdefghij

-

++++-++++ 0 123

0

I

2

3

‘a 30 “C+O

OC

30 “C (a)

30 OCcomdexes -20

+I

-10 I

I

10 I

I

20

30

I

I

A+C+U:

T TT TT 2e;fI;fl+c: T TTT TT TT T TTRTGTRGTRTRCTCTTTCTTCRRCARTTRRRTRCTCTCGGTRGCCflRGTTG RRTRCRTCATRTGRGRRRGRRGTTGTTRRTTTRTGRGRGCCRTCGGTTCRRC 2';R;R+C: t TT TT ttt R+C+U: TT TTt

30 OCto 0 OCcomplexes: -20

-10

I

+1

IO

20

30

I

I

I

I

I

TT T TT T TTT TT TT T TT TT T TTRTGTRGTRTRCTCTTTCTTCRRCRRTTRRRTRCTCTCGGTRGCCRRGTTG RRTRCRTCRTRTGRGRRRGRRGTTGTTRRTTTRTGRGRGCCRTCGGTTCRRC TT tt R: TT TT R+C: t R+C+U: tT TTt

R+C+U: R+C: R:

( b) Figure 6. Effect of initiating nucleotides on the stability of the transcription bubble upon shift t,o 0 1’. (a) Analysis of KMnO, hyperreactive sites on the non-transcribed strand. Plasmid pTZ2, 4 fmol 5’.end-labeled on the non-transcribed strand, was incubated with TFIIIC (17 fmol), TFIIIB (148 fmol) and pol III (2 fmol) for 40 min at, 21”(1. Buffer (lanes a and g). ATP (lanes c and h), ATP+CTP (lanes d and i) or ATP+(‘TP+ HTTP (lanes e and j) was t,hen added and incubated for 6 min at 30°C. Lanes a and f are controls without protein. Permanganate was added and incubated a further 1 min at 30°C (lanes f to j) or the reaction mixtures were first incubated for 6 min at O’Y’ prior to a 1 min treatment with KMnO, at 0°C (lanes a to e). (b) Map of the hyperreactive thymine residues in binary and initiat,ed complexes. Data for the non-transcribed (top) strand are derived from (a). Data for the transcribed (bottom) strand summarize the results from 4 experiments utilizing the same protocol as in (a). The lower-case t designates inconsistent and. on average, weaker, hyperreactivity to KMnO,. phase of repetitive initiation prior t’o abortive productive RNA chain elongation (Carpousis & Gralla, 1980; Luse & Jacob, 1987). We could, however, show that at least a subset of the ternary or complexes formed in the presence of ATP distinct from the binary (ATP+CTP) were complex. When pol III was allowed to form a 17-mer RNA chain at 30°C and then shifted to 0°C for six minutes, the T residues between base-pairs +6 and + 16 remained fully reactive to permanganate (Fig. 6(a), compare lanes e and j), whereas t,he binary complex rapidly lost T-reactivity at 0°C (Fig. 3(d); Fig. 6(a), compare lanes g and b). The pol

III-containing

ternary

complex

and CTP (i.e. arrested retained full T-reactivity lanes

i and

d).

Surprlsmgly,

formed

with

ATP

before nuclrotidr 6) also at 0°C’ (Fig. 6(a). c*omparr the

ternary

c*omplrs

formed with ATP displayed int,ermediate properties: T-reactivity at 0°C was lost between base-pairs - 5 and - 9. partially ret)ained from base-pairs - 3 to + 7. and the weak reactivity of T + 11 was full>retained (Fig. 6(a), compare lanes h and (a). It appears that t)he conformat,ion of t)hr non-t ran scribed strand in the binary complex formed al 21 “C or 3O”c’, and in the ternary complex that, results upon adding ATP and CTP was nearly iden-

RNA Polymerase I I I Transcription tical. Yet the latter predominantly contains nascent RNA chains of length three to five. (The nascent chains must be longer than 2 nt because the T-reactivities at 0°C of the ternary complexes formed with ATP alone and with ATP + CTP differ, and they cannot be longer than 5 nt because UTP is lacking: see Materials and Methods.) Some of us have noted previously that the reactivity of the transcribed strand to KMnO, is much weaker than that of the non-transcribed strand (Kassavetis et al., 1990). For reasons that we have not determined, the pattern of weak T-reactivity on this strand is variable at certain positions. Figure 6(b) (bottom strands) summarizes the results from four experiments that probed the transcribed strand with experimental protocols identical with that shown in Figure 6(a). Binary complexes formed and maintained at 30°C displayed invariant hyperreactivity to KMnO, at T +l, T +2, T +4 and T +5, and inconsistent hyperreaetivity (weaker, on average) at T -11, T +8, T +9 and T +lO (designated by lower-case t). Complexes initiated with ATP or ATP+CTP retained the T-reactivity pattern of the binary complex analyzed within the same experiment. Complexes initiated with ATP + CTP + UTP to form a ternary complex halted at nucleotide 17, lost reactivity at T - 11, T + 1 and T + 2, retained reactivity at T +4, T + 5, T + 8 and T + 9, and showed inconsistent reactivity at T + 10. Binary complexes shifted to 0°C displayed no T-hyperreactivity, whereas the ternary transcription complexes stalled at nucleotide 17 retained their T-reactivity pattern when shifted to O”C, as they did for the non-transcribed strand. Complexes initiated with ATP or ATP+CTP both lost the inconsistent reactivity at T + 8, T +9 and T + 10 at 0°C. Only (ATP + CTP)-initiated complexes retained inconsistent T-reactivity at T - 11. It, is known that the pol III of a ternary complex elongated to position 17 is resistant to attack by the polyanion heparin. The nascent RNA of such complexes can be quantitatively chased to fulllength by providing GTP (Kassavetis et al.. 1989). As expect,ed, the KMnO,-reactivity of T in the non-transcribed DNA strand of these transcription complexes was also insensitive to heparin (Fig. 7). Tn contrast, t#he complex formed at 25°C in the presence of ATP+CTP only was sensit’ive to heparin. The dist,inctive properties of initiated transcription complexes containing 3 to 5 nt and 17 nt RNA chains. respectively, suggest the existence of an intermediate state on the reaction path from the binary complex to the productively elongating ternary t)ranscription complex.

(b) Probing with

pal I I I-DNA complexes (OP),-Cd nuclease

the activity of chemical nuclease The 1 ,lO-phenant’hroline-Cu’ complex, (OP),-Cu’, acts from the minor groove to cleave DNA by oxidation of deoxyribose (Rigman & Spassky, 1989) at a rate that is determined by the conformation of the minor

0.20 0.00

53

Complexes

I

I IO

I

I 2.0

I

1 3-o

Duration of heporin treatmeni

I

I 40

(mm)

Figure 7. Resistance of initiated complexes to heparin sequestration. Plasmid pTZ1, 4 fmol 3’-end-labeled on the transcribed strand, was incubated with TFIIIC (14 fmol), TFIIIR (8-9 fmol), and pol III (2 fmol) for 40 min at 25°C. ATP+CTP (0) or ATP+CTP+UTP (m) was added to initiate transcription and incubated for 5 min. Heparin was then added to 250 pg/ml and the incubation continued for the time specified prior to the 30 s treatment with KM&,. The “zero minute” controls contain no heparin. Hyperreactivitg at T + 4 to T + 9 was quantified as described in Materials and Methods. The T-reactivity of t)he (A + C)-containing and (A + C + I’)-containing t.ernary transcription complexes was normalized at 0 min. (The ratio of absolute values of integrated (A+C) complexes was I : 2.3).

to (A+C+U)

complexes

film density for at, time

zero

groove. Alt’hough free single-stranded DNA reacts slowly with (OP),-Cu’, it has been shown that the unpaired transcribed strand of an open promoter is hypercomplex with E. coli RXA polymerase reactive to (OP)2-Cu’, presumably due to an interaction of this strand with the active site of RNA polymerase that favors (OP),-Cu’ binding (Spassky & Sigman. 1985). Figure 8 shows that this property of (OP),-Cu’ cleavage also holds for pol III transcription complexes. The aligned densitometric scans of Figure 8(a) compare the partial cleavage patterns of the transcribed strand of the naked SUP4 tRNA gene with a TFIII(C+ B)-DNA complex in the segment that contains the transcriptional start and the upstream DNA-binding site of TFTIIB. TFIIIB substantially protected the approximat,ely 30 contiguous base-pairs of upstream DNA from cleavage. Addition of pol III to the TFIII(C+ B)-DNA complex made the DNA between base-pairs + 3 and - 11 hypersensitive to cleavage by (OP),-Cu’ on the transcribed strand (Fig. 8(b)). The formation of the ternary complex arrested at nucleotide 17 eliminated the hyperreactivit,?; of the t,ranscribed strand bet,ween basepairs + 2 and - 11, and increased cleavage between base-pairs +3 and +6 (Fig. 8(c)). On the non-transcribed strand, TFIIIB protected a contiguous stretch of DNA from base-pairs -41 but see Fig. 8(d)). to - 13 (data not shown, Addition of pol III had no further effect on the (OP),-Cu’ cleavage pattern in the region surrounding the transcription start (Fig. 8(d)), nor did formation of ternary complexes arrested at posit’ion +17 (data not shown).

G. A. Kassavetis -

--

--

CfB c+B+pol III

C+E+pol ID +3NTP c+B+polm

+6H+3

(d --~ -

No protein C+B+polm

Figure 8. I’henanthroline-copper footprinting of transcription complexes at the transcriptional start and upstream sequence. Plasmid pTZ1, 2 fmol 3’-end-labeled on (a) to (c) the transcribed strand or (d) on the nontranscribed strand, was incubated with TFIIIC (17 fmol). TFIIIB (39 fmol) and pol III (2 fmol) for 40 min at 25”(‘, followed by the addition of ATP. CTP and UTP for 2 min before submiting the sample to the (OP),-Cu’ cleavage reaction for 30 s. as indicated. The autoradiogram was scanned and analyzed as specified in Materials and Methods. Densitometric scans of lanes that are compared in the individual panels were aligned and normalized to unprotected sequence.

(c) c’sing footprinting methods to search for an RXA component in the S. cerevisiae pal III transcription system

essential

It has been shown that a separable RNA component, TFIIIR, is required for efficient pol TIT transcription in a system derived from the domestic silk month Bombyx mori (Young et al., 1991). TFIIIR appears to be a general transcription factor in B. mori required for tRNA and 5 S RNA synthesis. Since the pol III transcription apparnti of higher and lower eukaryotes bear many similarities, it seems likely that an RNA component should also participate in S. cerevisiae pol III transcription. DNA footprinting methods offer a special advan-

et al.

tage in searching for such a component. because the associated footprint signature of each step in the formation of the transcription initiation complex, in and RNA cahain elongation promoter opening, removes the necessity of preserving the reaction product. RNA, while at t,he same time specifying the step in take reaction sequence at which an essential kanscription factor RNA might function We have therefore exploited footprinting methods and t,he available information about S. cerr?*isiap pol TIT transcription complexes to search for a nucleasrsensitive step in transcriptiona, initiation. Young et al. (1991) found t,hat incubat,ion of‘ a crude 11. mori nuclear extract in the presence of 192 units of micrococcal nuclease (LMNase) per milliliter for 30 minutes at 37°C’ prior to the addition of’ ECTA (to chelate (‘a’+ and t,hereby inactivate MNase) and template l>XA abolished pal TTI trailscription. Figure 9(a) shows the results of a similar. test of the effect on pol III transcription of’ incubating MNase wit’h highly purified TFTTIC. pal 11 I and partially purified-TFTTIB prior to addition ot EGTA and SUP4 tDNA. When TFTIIB, TFTlIC’ and pol ITT were incubated for 30 minutes at 21 7’ with 625 tjo 2500 unit)s of MNasejml (Fig. 9(a). lanes a to (8) in the absence of I)K ‘A ternpla,t,e. litftlr or no inhibition of transcription occurred cwnpared with a control incubated without MXase, although inctl~ bation at 210(’ did itself result in some inact#ivation of onr or more of the transcription components (not shown). For comparison. lane rl displays thtx accumulation of t’htt SUP4 tKh’A transcript after 60 rninutes of R&A synthesis in which nc) preincuhation of t,ranscription components oc*t:urred in the absence of DNA template. whereas RNA synthrhsis in lanes a to c occurred for 30 minut*es. Incubation of preformed SUP4 tRNA (lane d) with 625 t’o 2500 units of MNase/ml efficiently hydrolyzed this RNA (lanes v to g: although not’ shown, no sma,ll molecular mass products remained on the gel). The effects of high conc:ent,rat,ions of K%ases on the formation of transcription initiation c~iiplexrs and elongating t,ernary complexes was nrxt cbxanlined by probing t,he open l)N,L\ of t hr promotrlcomplex and t,he transcription bubble with KMnO,. TFTTTB, TFTIIC’ and pol ITT were incubated with 50 ,ug of RNase X/ml and 1400 units of RNasr T,jml (Fig. 9(b). lane c). or twice these (.(~ncr~lltrations (lane d) for 30 minutes at 21 “CI. followed by addition of thfl l)NA probe and ATP. C’TP and l’TP I o init,iat#e transcription and form a trrnar)c~ornplex iL1 position 17. Preincubat,ion with t,hese ribonu&a.srs had no effecat on the KMnO, foot.print relative to a buffer c*ontrol (lane 1)). Relatively minor ifla(*Gviltion of transc-ription c~omponrnts ocr*urrrd by l)rt’ incubation in the absence of l)SA frmpIa1~~ (compare lane b with the no-preincubation c*ontrol in lane v). The ability of RNases A and ‘I’, to digest RXA in the presence of TFIlIC. TFll I B and pal TI I is shown as a control. in lanes f’ to i of Figure 9(b). Labeled SYPd tRX*d (primary transcript,, mature tRNA and processing products; lane f) was incrrbated for 30 minutes at 21 7’ in t,he presrnc:tb of

RNA

0

Polymerase

I I I Transcription

bcdefg

SUP4 +

MNase.

ix 2x

4x

-

lx

2x

4x

(a) a

b

polEI:RNose A: RNose T,: -

+ -

Reoctw

c

d

e

fg

h

i

T {

+ + Ix 2K 1X2X

+ -

Complexes

55

TFIIIB, TFIIIC and pol III, without (lane h) and with 3 pg of RNase A/ml (lane g) or 1400 units of RNase T,/ml (lane i). Just 3 pg of RNase A/ml sufficed to hydrolyze all the RNA to small fragments under the experimental conditions of Figure 9(b) and 1400 units of RNase T,/ml hydrolyzed most of the RNA (all of the primary transcript and mature tRNA) into small fragments. In a separate experiment (not shown), TFIIIB, TFIIIC and pol III were incubated with 20,000 units of RNase T,/ml for 30 minutes at 21 “C prior to addition of the DNA probe and ATP, CTP and UTP to form a ternary complex at nucleotide 17. Preincubation with these larger amounts of RNase T, again had no effect on the KMnO, signal relative to the buffer control. We conclude that our S. rereoisiae-derived RNA polymerase III transcription system does not contain an RNA component that is both accessible to micrococcal nuclease digestion and necessary for any rate-limiting step in the formation of ternary transcription complexes.

4. Discussion

( b)

Figure 9. Searching

for a nuclease-sensitive component of the pol III transcription system. (a) Effect of preincubat,ing TFTTTC. TFTTIB and pol III with micrococcal nuclease (MXase) on the ability to synthesize tRNA. For lanes a to c. TFIIIC (17 fmol), TFIIIB (7.4 fmol) and pol ITT (3 fmol) were incubated with MrJase at 625 units/ml (lane a). 1250 units/ml (lane b) or 2500 units/ml (lane c) for 30 min at 21 “C. EGTA was added to 3.6 mM, 6 min later 100 ng of pTZ1 DKA was added, and all samples

were incubated

for 30 min prior

to a 30 min multiple

round transcription reaction. For lanes d to f, TFIIIC, TFIIIB and pol III were allowed to bind to pTZ1 for 36 min at 21 “C: labeled iXTPs were added for 30 min of transcription followed by the addition of MNase diluent (lane d). or Mh’ase at 625 units/ml (lane e), 1250 units/ml (lane f) or 2500 units/ml (lane g) for a further 30 min incubation. (b) Effect of preincubating TFIIIC, TFTIIB and pol ITT with R?u’ases A and T, on their ability to form ternary transcription complexes. TFIIIC (14 fmol). TFTIIB (I 1 fmol) and pol III (3.6 fmol) were incubated with BSA diluent (lane b), 5Opg RNase A/ml and 1400 units of Rh’ase T,/ml (lane c). 100 fig RP;ase A/ml and 2400 units of RBase T,/ml (lane d). or 4 fmol of pTZ1 DNA (lane e) for 30 min at 21 “C: 4 fmol of pTZ1 DNA (lanes b to d) or BSA diluent (lane e) was added and incubation continued for 30 min. ATP. CTP and UTP were added for 16 min to initiate transcription. The formation of ternary transcription complexes was assayed with a 30 s treatment with KMnO,. Lane (a) is identical with the lane (b) reaction, except that pol III was omitted. La.nes f to i: sensitivity of SUP4 tRNA to digestion by RNases A and T,. TFIIIB, TFIIIC. pol III and purified 32P-labeled SITP4 tRNA (synthesized with fraction BRa: Kassavetis et al.. 1989) were incubated for 30 min at 0°C (lane f) or at 21°C (lanes g to i) with BSA diluent (lanes f and h). 3 pg RNase A/ml (iane g); or 1400 units of Rh’asr T,/ml (lane i) and the products analyzed by gel electrophoresis.

RNA polymerase TII uses the TFIITB-DNA complex as its beacon for locating the start site of transcription. The placement of TFTTTB on a tRNA gene appears to be determined primarily by the location of the box A promoter element’, to which al., 1990). d TFTIIC binds (Kassavetis Photocrosslinking studies (Bartholomew et al., 1991: Kassavetis et al., 1991) indicate that the 135 kDa subunit of TFITIC protrudes upstream from the box A region of the SCP4 tRNA gene to assemble the 70 kDa component of TFTTIB onto its gene-proximal upstream binding region. The 90 kDa component of TFIIIB must then assemble onto the gene-distal portion of the upstream binding region in order for pol III t’o stably associate with the tRNA gene. Analysis of 71 9. cerwisinr tRXA genes indicates significant sequence conservation surrounding t’he transcriptional start, from basepairs -4 to +8: T H T C A w - A A A W W (unpublished data; H = not G. W = A or T: upper and lower-case letters refer to greater and lesser statistical significance, respectively). Tn prior work, replacing 5’-flanking sequence by deletion and substitution with vector sequence has yielded varying effects on S. cerevisiae tRNA synthesis; little effect’ (Koski et al., 1982) as well as pronounced inhibition (Raymond & Johnson, 1983; Raymond et nl., 1985) have been noted. In order to assess effects on t.ranscription that might be generated by altering DNA sequence at the transcriptional start, or placing TFIIIB at different upstream sequences, the steps involved in pol III binding and transcription initiation must be delineated. RNA polymerase IIT assembles onto a TFTIICTFTTIB-tDNA complex at the site of transcript’ional initiation at 0°C (Fig. 1). This association is quite stable, with little or no dissociation of pol III after six minutes of competition with polp(dI-dC). poly(dl-dC) (Fig. 2). Tn the 0°C binary complex, the

56

G. A. Kassavetis

DNA segment surrounding the transcriptional start) site appears to retain its duplex form, as evidenced by the low reactivity of T-residues t’o oxidation by KMnO, (Fig. 3(a)), contrasted with full sensitivity of T residues within the transcription bubble of the 17-mer ternary complex to KMnO, oxidation at 0 “C (Fig. 6(a)). This 0°C complex of pol III therefore mimics the closed promoter complex of E. coli RSA polymerase. However, while closed E. coli RNA polymerase-promoter complexes are generally in rapid equilibrium with the free enzyme, pol III in its closed SUP4 tRNA gene promoter complex is not. The degree to which the stability of the closed complex depends on pol III-TFIIIB. pol ITTTFIIIC and pol IIILDSA interactions (both sequence-specific and non-specific) is clearly a significant question that is now accessible to analysis. For example, if TFIIIB anchors pol III to the starts it must release before, or concurrently with. RNA chain elongation. The transition from the closed t’o the open promoter complex is a tempera,ture-dependent) process (Fig. 3(a). (b) and ((2)) and it) is rerersihle (Fig. 3(d)). Unlike t’he steep closed-to-open t’ransitions of strong E. coli promoters (Spassky et al.. 1985), localized melting at the transcriptional start increases J>rogressively from IO" to 40°C. In part. this broad temperature range may be due to the fact that the formation of the open DNA segment, is not, entirely co-operative: there is some indication that the T residues between -9 and -5 begin to melt out at a slightly lower temperature t,han T -3 to + 11 (Fig. 3(c)). For the approximately 14 bp downstream segment, the estimated AN&,, from Figure 3(c). 30 kcal/mol (1 cal = 4.184,J). is low for the number of hase-pairs involved (6 to 20 kcal/mol bp). and low for comparable processes at the E. coli lac CV5 promoter (120 kcal/mol; Spassky et al., 1985), which are thought to involve a compara~ble ttumber (14 to 22) of base-pairs (Straney & Crothers. 1987: Buckle & Kuc. 1989; Sasse-Dwight 8: (iralla. 1989). Explanat,ions for low AH0 might invoke less than full st,rand separation and compensating exot’hermic DNA-protein and protein-protein interactions. The apparently sharper thermal t’ransitions of transcrip t’ion activity in Figure .5 than of promoter, opening in Figure 3(c) can be qualitatively reconciled in terms of information contained in Figure 4, and the following standard, although simplified, scheme: pol TII f TFIII(H

+ C-DNA

Closed G step1 2” complex

11

step 2

l 7NTPsOpen 3” complex / step3 2” complex In this scheme. the format’ion of the stable ternary complex containing a 17 nt) RSA chain represents an essentially irreversible st’ep. The dat,a of Figure 4 specify that equilibrium for steps 1 and 2 together was nearly established within 20 minutes at 15°C. was relatively faster at higher temperatures and, by

et al.

extrapolation, should be quite slow at I IO (‘. Once equilibrium was achieved for open complex formation at 15”C, addition of ATP. (‘TP and L’TP resulted in fairly rapid formation of t,ernar?complexes halted at position + 17 (step 3: 1/2 I 45 s; data not shown). Thus t,hr rrlativrl~ rapid rate for step 3 to form an irre\.ersible t)ernar> complex at I.5 “c’ drives ternary complex formation t’o completion if suficient time is ,giveil for slower steps 1 and 2 to reach equilibrium. At I()“(‘, equilibrium was not, established for st’eps 1 and 2 at thth t,ime of sampling (extrapolated from Fig. 4) arld stcbp 3 also became somewhat limit,ing 2 approx. 2 min: data not) shown). That combination gent+ ated a hig t,emperature effect in Figurfa 5 on t Irtx formation of t)ernarp transcription c~otrrplt~xrs bet)ween 10” and 15“C. E’. r:oli RNA pol~merasr has bct~n shown to undergo a process of continuous abortivcb init’iat ion whrn rfVTPs arc. added to open promotrr (*on1 plexes, forming oligonuc,leotitles ranging iti sist> from ovvt’ th tlutnhf~r 01 2 to 9 nt in vast molar exwss open complexes (Carpousis 8.~(:t*alla. I9XO; (iralla r,t trl.. 1980). The tirnfb that an c~nxytn(~tnolrc~ulc sprnds reaction differs for differerii in this idling promoters and also depends on r’cactic~tr c~ontlitiot~s. The transition from t ht. rriterati1.c init id iota l)hast’ to productive RX:1 chain ~~longation is ;lc*c~ompat~i(‘(l by the rf~lt~asc of the 0” subunit of RNA polymerase (Hanson & Jlc(‘lurr. 1980: Krummrl & (‘hamberlin. 1989) and by changes itt the DIVasr 1 footprint (Stranry & (‘rothers, 1985: Krummrl 8 (‘hamberlin. 1989). RI\&4 l)olyrnt~rasc~ IT from humans likewise undergoes a pro(‘t’ss of’ abort ivct initiation at low c,ollc,c~ntrat,ioi~s of NTI’ (Lusr k ,Jacol), 1987). Ttl our work. t’he ntilizat’ion of WIW tively high concrntrations of NTP and the forma tion of only a few femt,omoles of iWtiVf t ratxwript~ion complex (with c>xtensively purified f’ractions of TFTITC’, TFTTTH and pol TIT), puts t trtht1irec.t search for. and analysis of. abortivrb initiation c-vfwi5 beyond our tea,c*h. If an abortivcl initiation exists for pal ITT transcriptiorl. it is c.fartainlj conceirable that it might rrprrserrt. ;I rate-limit,iny step fhr transc~ription of the small RNA-c*ocling pal ITI-sl)ecific genes. and t’hat it might br t~rgulat~ecl pither by pr~mar~y sclquenct’ around I hr t ranscaril,Gonal st#art or 1)~ the tightness of ‘t’l~‘lTIR~l~ol I1 I interac+ons. When pol TIT initiates tr;triswipi.ion in t,hr abscncfa of’ OIW of the t’our rSTPs to forrtj :I t,ernary transcription complex cwntaitling ii rtaswtrl RNA chain of 10 nt (5 S rR,S,jS.genras). 12 nt. (Sl’l’ci tR,NATy’) or Ii rtt (SITI’ tjlZSATy’)- this c*omplrs is highly rrsistant to disruption by thr polyailiorrs heparin and poly(dC). to 0.5 M-NAY. or 0.3”),, (w/v) Sarkosyl (Fig. 7: Braun nl.. 1989: Kassal-&s u/.. 1989. 1990: and datja not shown). Addition of t hf, missing irnolfwticlf: rapidly and cluarit ii,iltivt~l> chases t hrse stable c~omplexes in10 filll-length products (Iia.ssilvrt,is d.. 1989; +Xtltl diitil 1101 shown). demonstrating that t’hr?- i1t.e l)ro(lnc+ivc~. rather t’han abortive. Since pal 111 irr t,he bittar~open promoter complrs rapidly cblost,supon shifting t

t

(t,

IltliiSt’

cd

Pt

d

RNA Polymerase III to 0°C (Fig. 3(d)), it was possible to distinguish pol III ternary transcript’ion complexes that were limited to extending an RNA chain to 2 nt (ATP only) and 5 nt (ATP + CTP) from binary complexes by probing with KM&, (Fig. 6). RNA polymerase III allowed to synthesize the first 5 nt displayed little evidence for an advance of the transcription bubble, retaining hyperreactivity to KMnO, at T - 11 and slightly increasing reactivity at T + 11. Its heparin-sensitivity also distinguished this complex from pol III engaged in productive RNA chain elongation (Fig. 7). Upon extending the RNA chain from (a maximum of) 5 nt to 17 nt, the size of the t’ranscription bubble apparently diminished from 22 nt to between 13 and 17 nt (Fig. 6(b)). (The range for each estimate is determined by the distribution of T in the sequence.) Our current estimate of t,he rxt’ent of DEA strand opening in the binary complex (22 nt) differs from our previous estimate (14 nt; Kassavetis et al., 1990) due to inclusion of T -9, T - 11 and T + 11 (which were obscured by a high background in the previous work). In summary, the presented data indicate t,he formation of at least two distinct post-initiation ternary transcription complexes. different in their extent of RNA chain elongation. LVe have noted previously t’hat KMnO, reactivity on the transcribed strand is significantly weaker than on the non-transcribed strand (Kassavetis it al., 1990). In the 17-mer complex, T + 10 and T + 12 on the t’ranscribed strand failed to react wit’h KMnO,. whereas T residues + 3, +4, + 8 and +9 did react. We suggested that this may directly reflect the extent of the RNA-DSA hybrid uithin the transcription bubble; direct interaction of pol III with the non-base-paired thymine may also result in protection. When pol III is allowed to initiate with XTP alone and (presumably) form nascent) pppApA, the stretch of reactive T residues in the -9 to T + 11 segment of the non-transcribed st’rand of the open complex at 30°C shrinks leaving only T -3 to + 11 reactive at 0°C (Fig. 6(a) and (1))). The preferent’ial loss of reactivity T -9 to T -5 support’s t’he notion that this segment may melt independently of the -3 to + 11 region. as suggested in discussing the temperature-dependence of promoter opening (Fig. 3(c)). Within the -3 to + 11 bubble. only T + 1 and T +2 on the transcribed strand remain consistently reactive to KMnO, where one would expect’ T residues + 3, + 4. + 8, +9 and + 10 also t)o be consistently reactive (Fig. 6(b)). In fact. T +3 and T +4 in the r\TP-initiated complex at 0°C were reactive in only one of four experiments and T residues + 8, + 9 and + 10 were never reactive at 0°C in either t,he ATP or ternary complex. The (ATP + CTP)-initiated simplest not’ion is that t,hese unreactive or inconsistently reactive T residues are protected by interaction with pal 111. The hyperreactivity of the template strand within the single-stranded transcription bubble between base-pairs - 11 and + 3 to cleavage by (OP),-Cu’ (Fig. 8) supports this view. is normally resistant to Single-stranded DNA

Transcription

Complexes

57

(OP),-Cu’ cleavage. The ability of the template strand to be cleaved with the open promoter transcription bubble could be due to binding of (OP),-Cu’ to RNA polymerase within close proximity of the template strand; alternatively, the interaction of the template strand with RNA polymerase may constrain stacking interactions with (OP),-Cu’ et al., 1990). (Thederahn It is useful to compare the transcription cycles of pol III and E. coli RNA polymerase in order to discern evolutionarily conserved steps; in this pathway. The comparison begins with TFIIIB and 0” both of which interact with DNA sequences directly upstream from the t’ranscriptional start, and act catalytically to direct REA polymerase to this site. Both pol III and E. coli RNA polymerase initially form a closed complex in which the DPiA duplex remains intact but, whereas the pol IIIDNA closed complex is relatively stable, its prokaryotic counterpart is not. Transition from the closed to open complex is an endothermic process for each polymerase, resulting in the unwinding of 14 t’o 22 bp of DNA. Both polymerases have two forms of post-initiation ternary complexes: those with short. init’ial RNA chains (generall:y < 10 nt) are characterized by reiterative init,iation in prokaryotes. while those with longer RX.4 chains are characterized by great st’ability and resistance to dissociation by polyanions and high ionic strength. A new pol III factor: TFIIIR, consisting of RXA and free of protein, has been found to be essential in an in U&O transcription system derived from 1991). Evidence was B. mori (Young et al., presented that this factor has a direct role in pol III transcription rather than one of count)eracting an inhibitor in one of the other relativrlv crude fractions. In the case of t’he pol III transcription system from S. cwevisiae, pret’reatment of highly purified TFIIIC. pol III and extensively purified TFIIIB with MNase has no effect on any rate-limiting step of the transcription cycle (Fig. 9(a)). Likewise, pretreatment of TFIIIB. TFIIIC and pol III with RNase A and R?;ase T, does not prevent pol III from binding to the SUP4 tRKA gene and productively initiating transcription (Fig. S(b)). The simplest interpretation is that there is no essential pol III R8?2A component in the in vitro 9. cer&siae transcription system. The requirement for an essential RXA component in B. fmori may: for example, correlate with the putative substitution of the unitary yeast TFIIIC multi-protein assembly by two separable B. mori factors, TFIII(‘ and TFIIID (Ot’tonello et al., 1987), whose assembly on the DSA template might require a structural RNA. Alternatively, if a structural RNA is required for yeast’ pol III transcription, it might) be so embedded m one of the transcription factors that the crucial portions of it are inaccessible to nucleases. In this alternative. the structural integrity of the large and complex TFIIIC might be contributed by a structural RNA. Final resolution of this problem may require sensitive measurement of RSA content in highly purified fractions, coupled with an accurate

58

G. A. Kassavetis

counting pal TIT.

of active

molecules

of TFIIIB,

TFTIIC

and

We thank B. R. Braun and (1. Bardeleben for helpful comments on the manuscript. Our research was support,ed by a grant from t’he NIGMS. References Bartholomew. B., Kassavetis. G. A. & Geiduschek, E. P. (1991). Two components of S. cerevisiae TFIIIB are stereospecifically located upstream of a tRNA gene and interact with the second largest’ subunit of TFIIIC. Mol. Cell. BioZ. 11. 5181-5189. Braun. B. R,.. Riggs. D. L, Kassavetis. G. A. & Geiduschek, E. P. (1989). Multiple states of proteinDPU’A interaction in the assembly of transcription complexes on SaccharomycPs cerezlrsiae .5S ribosomal RR’A genes. Proc. Nat. Acad. Sci., U.S.A. 86. 2530-2534. But. H. (1987). Initiation of prokaryotic transcriptionkinetic and structural approaches. In Xucl~ic Acids and &foZecuZar Biology (Eckstein, F. & Lilley, D. M. ,J.. eds). vol. 1. pp. 186-195. Springer-Verlag, Berlin. Buckle. M. & But. H. (1989). Fine mapping of D?u’A single-stranded regions using base-specific chemical probes: study of an open complex formed between promoter. RNA polymerase and the Zac IV5 Biochemistry,

28, 4388-4396.

Carpousis. A. tJ. $ &alla. .J. 1). (1980). Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in Gtro at t’he Zac UV5 promoter. Biochemistry, 19. 3245-3253. Gabrielsen. 0. S. & Sentenar. A. (1991). RPjA polymerasr III(C) and its transcription factors. Trends Biochem. Sri. 16. 412-416. Geiduschek. E. P. & Kassavet,is. G. A. (1992). R’XA complexes. In ITT transcription polymerase Transcriptional Regulation (McKnight, S. 1,. 8: Yamamoto; K. I~., eds). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, IW. In the press. Gill, 8. (‘., Wager, T. D. & von Hippel, P. H. (1990). Thermodynamic analysis of the transcription cycle in E. coli.

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Gralla. ,I. D., Carpousis. A. tJ. & Stefano. ,J. E. (1980). Productive and abortive initiation of transcript,ion irt dro at the Zac IV5 promoter. Biochemistry, 19. 5864-5869. Hansen, U. M. & McClure, W. R. (1980). Role of the o subunit of Escherichia coli RrU’A polymerase in initiabion. J. Biol. Chem. 255, 9556-9563. Hayatsu, H. $ I:kita; T. (1967). The selective degradation of pyrimidines in nucleic acids by permanganatr oxidation. Biochem. Biophys. Ren. Commm. 29. 556-56 1. Kassavetis. (:. A., Riggs, 1). L.. Kegri, R.. Nguyen. I,. H. & Griduschek, E. P. (1989). Transcription fact’or TIIB generates extended DNA interactions in RXA polymerase III transcription complexes on tRh’A genes. Mol. CeZl. BioZ. 9. 2551.--2566. Kassavet’is, G. A.. Braun. B. R.? Nguyen, I,. H. & Geiduschek. E. P. (1990). S. cerevisiae TFIIIB is the transcription initiation factor proper of RNA I~olymerase TII, while TFTIIA and TFITIC are assembly factors. Cell, 60. 235-245. Kassavetis. G. A.. Bartholomew. B.. Blanco, ,J. A.. Johnson. T. E. &r Geiduschek, E. I’. (1991). Two essential components of the Saccharomyces cerecisiae factor IIIB: transcription and transcription

et al

DNA-binding properties. t’roc. .V&. =Icnd. Sci.. [‘.1)‘.,4. 88. 7308-7312. Koski, R. A., Allison. 1). S.. Worthington. 11. & Hall. B. D. (1982). An in vitro RXA polymerase III system from S. cerevisiae: effects of deletions and point mutations upon SIP4 gene transcription. :1-ucl. ilcids Re.s. 10. 8127-8143. Krummel. B. & (‘hamberlin. M. tJ. (1989). RXA chain initiation by Escherichia coli RNA polymerase. St,ructural transitions of the enzyme in early ternary complexes. Biochewtiatry, 28. 782997842. Luse, D. S. & Jacob. U. A. (1987). Ahortivr initiation b> RXA polymrrase 11 in cdro at the adenovirus 2 major late promoter. .J. Biol. (Them. 262. 14990-34997. Mc(‘lure. W. R. (1985). Mechanism and caontrol of traninitiation in prokaryotes. Annu. Ilet%. scription Biochem. 54. 17 l-204. Ottonello. S.. Rivier. I). H., I)oolittlr. (:. )I.. Young. 1,. S. 8r Sprague. K. I’. (1987). The properties of a new polymerase III transcription factor reveal t,hat transcription complexes can assemble by more than one pathwav. EXBO .J. 6. 1921-1927. Raymond, (i. ,J. & .Johnson. ,I I). ( 1983). The role of non coding DS,A sequences in transcription and Sucl. .-lcids Rrs. 11. processing of yea,st tRsA. 5969-5988. Raymond. K. C‘.. Etaymond. f:. .I. C’ ~Jc~hnson. .J. 1). (1985). In Gv modulation of yeast tRR;A gene expression by 5’-flanking sequences. F:MHO .J. 4. 2649.-26.56. Sasse-Dwight,. S. B (iralla. .I. 1). (1989). KMnO, as a prohe for lac promoter DNA melting and mechanism in v!itro.

.I. Biol.