Inhibition of RNA Polymerase III Elongation by a
T10 Peptide Nucleic Acid*
Giorgio
Dieci
§,
Roberto
Corradini¶,
Stefano
Sforza¶,
Rosangela
Marchelli¶, and
Simone
Ottonello
§
From the
Istituto di Scienze Biochimiche and ¶ Dipartimento
di Chimica Organica e Industriale, Università di Parma, I-43100
Parma, Italy
Received for publication, October 13, 2000, and in revised form, November 2, 2000
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ABSTRACT |
The terminator elements of eukaryotic class III
genes strongly contribute to overall transcription efficiency by
allowing fast RNA polymerase III (pol III) recycling. Being constituted by a run of thymidine residues on the coding strand (a poly(dA) tract
on the transcribed strand), pol III terminators are expected to form
highly stable triple-helix complexes with oligothymine peptide nucleic
acids (PNAs). We analyzed the effect of a T10 PNA on
in vitro transcription of three yeast class III genes
(coding for two different tRNAs and the U6 small nuclear RNA)
having termination signals of at least ten T residues. At nanomolar
concentrations, the PNA almost completely inhibited transcription of
supercoiled, but not linearized, templates in a sequence-specific
manner. The total RNA output of the first transcription cycle was not
affected by PNA concentrations strongly inhibiting multiple round
transcription. Thus, an impairment of pol III recycling fully accounts
for the observed inhibition. As revealed by the size and the state
(free or transcription complex-associated) of the RNAs produced in
PNA-inhibited reactions, pol III is "roadblocked" by the DNA-PNA
adduct before reaching the terminator region. On different templates,
the distance between the active site and the leading edge of the
arrested polymerase ranged from 10 to 20 base pairs. Given their
ability to efficiently block pol III elongation, oligothymine PNAs lend
themselves as potential cell growth inhibitors interfering with
eukaryotic class III gene transcription.
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INTRODUCTION |
High throughput transcription of eukaryotic genes encoding small
RNAs involved in protein synthesis (i.e. the tRNAs and the 5 S rRNA) is one of the main tasks of RNA polymerase III (pol III)1 and its associated
factors (TFIIIA, TFIIIC, and TFIIIB). TFIIIA (a 5 S-specific component)
and TFIIIC are sequence-specific DNA-binding proteins that recognize
the internal control regions of class III genes. Once bound, they
direct the assembly of TFIIIB upstream to the transcription start site;
TFIIIB then recruits pol III and actively participates in the
transcription initiation step (1, 2). To satisfy the high cellular need
for its RNA products, the pol III transcription machinery maximizes the
efficiency of reinitiation in two ways: once formed, a functional
preinitiation complex is stably maintained at the promoter for multiple
rounds of pol III recruitment by the initiation factor TFIIIB (1); in
addition, a promoter-engaged polymerase initiates new transcription cycles more rapidly than the first one by means of a
terminator-dependent, fast recycling pathway (3). The
terminator of pol III-transcribed genes, generally a run of 5 or more
thymidine residues on the coding strand, can thus be viewed as a key
promoter element that, by allowing pol III to enter a facilitated
initiation pathway, greatly increases the overall transcription output.
Shortening or deleting the terminator element causes a transcription
impairment due to pol III readthrough (4) and also precludes the
entrapment of terminating pol III molecules for structural and
functional studies. Therefore, a nondestructive approach, such as the
protein roadblock strategy utilized for Escherichia coli RNA
polymerase (5), has to be used for the functional dissection of the
coupled termination/reinitiation processes. Peptide nucleic acids
(PNAs), recently developed nucleic acid mimics in which the nucleobases are attached to a pseudopeptide backbone, can form extremely stable complexes with complementary RNA and DNA targets and have thus the
potential to interfere with key steps of gene expression (6). In
particular, oligopyrimidine PNAs bind with high affinity to double-stranded DNA targets through a strand displacement mechanism, in
which two invading PNAs form a Watson-Crick-Hogsteen triple-helix with
the complementary DNA strand, leaving the other strand unpaired (7).
The potential of oligopyrimidine PNAs for gene-targeting strategies has
originally been documented in studies demonstrating the
PNA-dependent arrest of transcription elongation by phage RNA polymerases and human RNA polymerase II on artiïficial DNA templates bearing an oligopurine cassette on the transcribed strand (8-11). We reasoned that the poly(dA) tract present on the transcribed strand of class III gene terminators represents a unique natural case
of a widespread eukaryotic control element that can be targeted by
oligothymine PNA molecules. To gain insight into the structural and
functional features of a late elongating pol III and to lay the grounds
for a detailed understanding of the anti-gene properties of PNAs, we
thus set out to analyze the transcriptional effect of a
terminator-bound oligothymine PNA in a well-defined in vitro pol III system from the yeast Saccharomyces cerevisiae.
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EXPERIMENTAL PROCEDURES |
PNAs--
The
H-T10-D-Lys-NH2 PNA was synthesized
manually, according to published procedures (12-14), on a
(4-methylbenhydryl)amine resin (Novabiochem) in a 6-µmol
scale. Boc-D-Lys(2-Cl-Z) (Novabiochem) and Boc
-T PNA
monomers (Perseptive Biosystems) were used. Coupling reactions were
carried out using
O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate as condensing agent and
N,N-diethylcyclohexylamine as base, with a 4-fold
molar excess of the PNA monomer. A trifluoroacetic acid
(TFA):trifluoromethanesulfonic acid mixture was used to release the
T10 oligomer from the resin. The product was precipitated with diethyl ether and purified by reversed phase high pressure liquid
chromatography on a Radialpack C18 column (Waters) with the following
gradient: 100% A (H2O:CH3CN:TFA = 1000:25:1) for 3 min, then a 22-min linear gradient to 100% B
(H2O:CH3CN:TFA = 25:1000:1) at a flow rate
of 2 ml/min. The product was isolated (retention time: 10.6 min) and
identified by mass spectrometry. Electrospray ionization - mass
spectrometry: 1404.5 (MH22+), 936.7 (MH33+); estimated mass for
MH+: 2807.1, calculated mass: 2807. The synthesis of the
PR11 PNA (H-GTAGATCACT-NH2) has been described recently
(15). Stock solutions of the PNAs were prepared in double-distilled
water. PNA concentrations were determined spectrophotometrically using
the following
260 (M
1
cm
1) values for the different nucleobases: T, 8800; C,
7300; A, 10,400; G, 11,700.
Transcription Templates--
Plasmid-borne class III genes were
used throughout this study. The yeast tRNAIle(UAU) and
tRNAPro(UGG) genes, identified as I{TAT}LR1 and
P{TGG}FL, respectively, by the MIPS nomenclature found on
the web, were polymerase chain reaction-amplified and inserted into the
pBlueScript KS(+) vector as described previously (16). Sequence
analysis of the tRNAIle gene revealed a terminator element
made up of 12 T residues, instead of the expected 10. The
SUP4 tRNATyr gene (4), in the pRS316 vector, was
a gift of Salam Shaaban. The tRNAGlu(UUC) gene, in the
pUCGlu plasmid (17), is identified at MIPS as E{TTC}ER2. The yeast
SNR6 gene was carried by plasmid pB6 (18). The
tRNAAsn(GUU) gene (referred to as N{GTT}CR at MIPS),
with 65 bp of 5'- and 84 bp of 3'-flanking regions, was amplified from
yeast genomic DNA using Deep Vent DNA polymerase (New England BioLabs)
and the following oligonucleotide primers: Asn_gtt19 (forward),
5'-CATACTCGAAGGGTAGTTGG; Asn_gtt19 (reverse),
5'-GATTTTTCCATTCGCCATGC; the resulting amplification product (235 bp)
was sequence-verified and inserted into the SmaI site of the
pBlueScript KS(+) vector.
Permanganate Probing--
The
pBlueScript-tDNAIle(TAT) plasmid (100 ng) was preincubated
with the T10 PNA in 1 mM Tris/HCl (pH 7.8) at
37 °C for 45 min in a volume of 11 µl, then permanganate
oxidation, piperidine cleavage, and cleaved DNA purification were
performed as described (19). Purified DNA samples were analyzed by
primer extension in reaction mixtures containing 0.25 mM
each of dATP, dCTP, dGTP, and dTTP, 25 mM Tris/HCl, pH 9.5, 5 mM MgCl2, 8 units of ThermoSequenase DNA
polymerase (Amersham Pharmacia Biotech), and 1 pmol of a 5'-end-labeled oligonucleotide primer (5'-CAGTCTACAATACAAAATTAGG) complementary to the
coding strand starting 67 bp downstream of the terminator element.
Primer extension reactions consisted of 31 cycles of denaturation
(94 °C, 4 min in cycle 1, 45 min in cycles 2-31), annealing
(50 °C, 45 min), and elongation (72 °C, 45 min); the resulting
samples were ethanol-precipitated, resuspended in formamide loading
buffer, and analyzed on a 5% polyacrylamide/7 M urea
sequencing gel followed by autoradiography.
In Vitro Transcription--
In all transcription experiments,
plasmid DNA was preincubated with the desired PNA concentration as for
permanganate probing. Multiple round or single round transcription
assays were then carried out as described (16), except for a reduction
of the final KCl concentration from 100 to 50 mM to
minimize PNA dissociation. Gel-filtration analysis of transcription
products on Sepharose 2B (Amersham Pharmacia Biotech) was performed as
described (20). Transcripts were quantified with the Multi-Analyst/PC
software (Bio-Rad) using phosphorimaging of dried gels obtained with a Personal Imager FX (Bio-Rad).
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RESULTS |
Binding of the T10 PNA to the tRNAIle Gene
Terminator--
Oligothymine PNAs with less than 10 T residues only
partially arrest transcription elongation by phage RNA polymerases
(10). We thus decided to use as a DNA ligand an
H-T10-D-Lys-NH2 PNA (hereafter
referred to as T10 PNA; Fig.
1A), in which the
D-lysine at the C terminus is known to favor DNA binding
(21) and the formation of right-handed helices (22, 23). A recently
characterized yeast tRNAIle gene bearing a T12
termination sequence was initially chosen as a target template (16).
PNA binding to the tDNAIle terminator was analyzed by
permanganate probing of the thymidine residues displaced by PNA
invasion of the duplex DNA (Fig. 1B). Because the tDNA was
carried on a closed circular plasmid, permanganate-induced cleavage
sites were detected as runoff products of primer extension reactions
initiated from a 32P-labeled primer annealing with the
coding strand downstream of the terminator sequence (19). As revealed
by the data reported in Fig. 1B, an increased cleavage,
specifically localized to the terminator region, was already evident at
200 nM PNA (lane 1) and became saturated at a
PNA concentration of 1.5 µM (lane 4). A
schematic representation of the (PNA)2-tDNA adduct is shown in Fig. 1C.

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Fig. 1.
Binding of the T10 PNA to the
tRNAIle gene terminator. A, structure of
the H-T10-D-Lys-NH2
(T10) PNA. B, permanganate probing of
T10 PNA binding. Unpaired thymine residues in the
terminator region of the tRNAIle(UAU) gene were monitored
by KMnO4 probing and primer extension after incubation of
the tDNA-containing plasmid with the indicated concentrations of
T10 PNA (left panel, top). The
right panel shows the results of a sequencing reaction
primed with the same oligonucleotide used for primer extension. The
positions of the B-block and of the terminator element are indicated on
the right. C, schematic representation of the
(T10 PNA)2-tDNAIle adduct; the two
internal control regions (A-block and B-block)
recognized by TFIIIC are indicated in gray.
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Sequence-specific Inhibition of tRNA Gene Transcription by the
T10 PNA--
To analyze the transcriptional effect of the
T10 PNA, the tDNAIle-containing plasmid was
preincubated with the PNA under low salt conditions, to favor strand
invasion, and then transferred to transcription buffer containing the
NTPs, pol III, and all the other protein components required to
reconstitute in vitro transcription. In the experiment
reported in Fig. 2A, the
tRNAIle gene was preincubated with increasing
concentrations of either the T10 PNA (lanes
1-7), a control PNA of unrelated sequence (PR11, lanes 8-14), or a standard T10 oligonucleotide
(lanes 15-18); transcription components were then added,
and multiple rounds of transcription (about 10 cycles) were allowed to
take place. A dramatic inhibition of transcription was observed in the
presence of the T10 PNA, with a half inhibitory
concentration of ~100 nM and a 90% inhibition at 200 nM. By comparison, no significant inhibition was produced
by the control PNA, nor by the T10 oligonucleotide. As
shown in Fig. 2A (lanes 5-7), inhibitory
concentrations of the T10 PNA caused the appearance of a
main shortened transcript. At maximally inhibitory PNA concentrations
(lanes 6, 7), the levels of this incomplete
transcript reached those of the full-length product; both transcripts
were detectable in low amounts (~5% of the output of the uninhibited
reaction) up to a T10 PNA concentration of 2 µM (not shown). A third, much less abundant transcript of intermediate size is barely detectable in lanes 6 and
7, but it was more evident in other experiments (see for
example Fig. 4A, lane 2).

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Fig. 2.
Transcription inhibition by the
T10 PNA. A, the
tDNAIle(TAT)-containing plasmid was preincubated with the
indicated concentrations of either the T10 PNA (lanes
1-7), a mixed sequence control PNA (PR11, lanes
8-14), or a T10 oligonucleotide (lanes
15-18). Transcriptional components were then added, and multiple
rounds of transcription were allowed to proceed. The migration position
of the tDNAIle primary transcript is indicated by a
arrowhead. The results of transcript quantification are
graphically reported below. B, the T10 PNA, at
the indicated concentrations, was incubated with yeast tRNA genes
having either a T8 (tDNAPro(TGG), lanes
1-5), a T6 (tDNAGlu(TTC), lanes
6-9), or a T7 (tDNA SUP4, lanes
10-13) terminator element, and transcription was allowed to
proceed as in panel A. The results of transcript
quantification are graphically reported below; transcription levels are
expressed as relative values with respect to the maximum
transcriptional output measured in each set of reactions.
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When genes with shorter terminator elements (6-8 T residues) were used
in place of the tRNAIle gene, the T10 PNA was
less effective in inhibiting transcription. As shown in Fig.
2B, a significant inhibition could still be observed in the
case of a tDNAPro(TGG) with a T8 terminator
(lanes 1-5), whereas only a 40% inhibition at 700 nM PNA was observed with the SUP4 tRNA gene,
having a T7GT6 terminator (lanes
10-13), and no inhibition was detected in the case of the
T6 terminator of the tRNAGlu(TTC) gene
(lanes 6-9). Along with the results of permanganate probing
experiments (Fig. 1), these data indicate that the T10 PNA
selectively binds to, and inhibits the transcription of, tRNA genes
with a terminator element made up of at least 8 consecutive T residues.
Effect of Template Supercoiling on PNA Inhibition--
The
T10 PNA half-inhibitory concentration measured in the case
of the tRNAIle gene (100 nM) is 5-10 times
lower than that determined in previous studies using templates
containing an artificially positioned T10 cassette to
analyze the PNA-induced arrest of phage RNA polymerases (10) and human
RNA polymerase II (8). These studies, however, were all conducted with
linearized DNA templates (to allow for runoff transcription), whereas
the tRNA genes utilized in our experiments were all in the form of
negatively supercoiled plasmids. An enhanced binding of PNAs to
supercoiled DNA has been reported previously (19). The plasmid
containing the tRNAIle gene was thus linearized by
restriction digestion and used to test whether template supercoiling is
responsible for the higher sensitivity of tRNA gene transcription to
PNA inhibition. As shown in Fig. 3, this
appears to be the case, because switching from a supercoiled
(lanes 1-4) to a linearized (lanes 5-8)
template caused a ~10-fold increase (from 100 nM to 1 µM) in the half-inhibitory concentration of the
T10 PNA.

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Fig. 3.
Effect of template supercoiling on PNA
inhibition. The tDNAIle(TAT)-containing plasmid, in a
supercoiled form (lanes 1-4, supercoiled) or
after linearization with EcoRI (lanes 5-8,
linear) was preincubated with the indicated concentrations
of the T10 PNA, then multiple rounds of transcription were
allowed to take place. The migration position of the tDNA primary
transcript (pre tRNAIle) is indicated on the
right.
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Mechanism of Transcriptional Inhibition by the T10
PNA--
The mechanism of PNA inhibition was investigated by limiting
transcription of preformed PNA·tDNAIle complexes
to a single round, a condition that by preventing reinitiation should
allow to monitor the effect of the PNA on the initiation and elongation
steps of the first transcription cycle. Reported in Fig.
4A are the results of an
experiment comparing the effect of the T10 PNA on either
multiple rounds (8 cycles; lanes 1 and 2) or a
single round (lanes 3-6) of tRNAIle gene
transcription. The PNA reduced the total output of multiple round
transcription to levels comparable to those of a single round reaction
(cf. lanes 2 and 3). Interestingly,
however, it did not appreciably affect the total amount, but rather the
size of the products of single round transcription reactions, with the
appearance of two shorter transcripts at a saturating PNA concentration
(lane 6). The PNA thus appears to arrest elongating pol III
before it enters the terminator region, thereby strongly inhibiting
subsequent recycling. Closer inspection of lanes 2 and
6 in Fig. 4A shows that, although very drastic,
the PNA inhibition of recycling is not complete. In fact, although the
amounts of the incomplete transcripts produced under multiple-round
(lane 2) and single-round (lane 6) conditions at
200 nM PNA are roughly the same, the full-length transcript
is more abundant in the PNA-inhibited multiple-round reaction
(lane 2), as if some residual reinitiation took place on a
few PNA-free templates. The fact that the shortened transcripts are
produced in the first round of PNA-inhibited transcription rules out
the possibility that they are synthesized by a second, colliding pol
III molecule. Moreover, no delay in the appearance of these shortened
transcripts, with respect to the full-length product, was revealed by a
time-course analysis of single round, PNA-inhibited reactions (data not
shown), thus suggesting that they do not originate from pol
III-catalyzed cleavage of a longer precursor (24, 25). The physical
state of the transcripts produced in PNA-inhibited reactions was next
investigated by molecular filtration on Sepharose-2B, a gel-permeation
matrix that can resolve unbound RNAs from RNA molecules that are part
of arrested ternary complexes. As shown in Fig. 4B,
full-length RNAs produced in a PNA-inhibited, multiple-round reaction
behaved as free, released transcripts (fractions 13-17), whereas
incomplete transcripts (the shortest one corresponding to ~90% of
the total) eluted in the void volume (fractions 7-11), as expected for
unreleased RNAs that are part of large, PNA-arrested ternary complexes.
A terminator-bound PNA thus acts as a roadblock for elongating pol III,
in such a way that the polymerization site of the enzyme becomes
arrested at a discrete distance from the upstream border of the
(T10 PNA)2-DNA adduct.

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Fig. 4.
Single-round transcription analysis of
T10 PNA inhibition. A, the
tDNAIle(TAT)-containing plasmid was preincubated with the
indicated concentrations of T10 PNA, and stalled elongation
complexes, containing a 7-nt long nascent RNA, were allowed to form by
the omission of CTP from the reaction mixture. Transcription was then
resumed by the addition of CTP, either alone to allow for multiple
rounds of transcription (lanes 1-2, MR) or
together with heparin (200 µg/ml) to limit transcription to a single
round (lanes 3-6, SR). The output of individual
reactions, relative to the output of the uninhibited multiple-round
reaction of lane 1 (arbitrarily set to 100) is indicated
below each lane (Txn). The migration position of the
full-length pre tRNAIle is indicated on the
left. The position of the most abundant shortened transcript
is marked by an asterisk. B, a scaled-up
transcription reaction (programmed with tDNAIle(TAT)
previously incubated with 400 nM T10 PNA) was
blocked with 20 mM EDTA, then loaded onto a 1-ml
Sepharose-2B column. Twenty fractions (50 µl each) were collected,
and transcripts contained in the indicated fractions (6-17)
were analyzed by gel-electrophoresis; an aliquot of the unfractionated
mixture was analyzed in parallel (input). The migration
positions of full-length and arrested transcripts are indicated on the
left.
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PNA-induced Transcription Arrest on Different Class III
Genes--
The size of the main unreleased product of PNA-inhibited
tRNAIle gene transcription was estimated by comparison with
two reference transcripts: the 150-nt long, full-length
pre-tRNAIle transcript (whose size was estimated on the
assumption that 5 U residues are incorporated before actual termination
(26)) and the 130-nt runoff transcription product of the same gene cut at an XhoI site located between the B-block and the
terminator. Because of the loss of the terminator, and thus of the fast
recycling pathway (3), the synthesis of this transcript was totally
insensitive to PNA inhibition (Fig.
5A, lanes 3 and
4) and much less efficient than correctly terminated
transcription (cf. lanes 1 and 3).
From the gel reported in Fig. 5A, a length of ~125 nt
could be calculated for the incomplete transcript associated to
PNA-arrested complexes. Fig. 5B illustrates the PNA-induced
transcription blockage observed with two other class III genes, both
containing a terminator element made up of 10 or more T residues. In
the case of the SNR6 gene, which codes for the yeast
U6 small nuclear RNA and has a T10 terminator, the
addition of the T10 PNA (100 nM) caused an 80%
inhibition of transcription and the appearance of an ~15-nt-shortened
transcript (marked by an asterisk in lane 4). At
variance with the residual, single-round transcription observed with
the tRNAIle gene (lane 2), the T10
PNA at a 500 nM concentration completely inhibited
SNR6 transcription (lane 6). This is most likely
due to weak PNA binding to the T7 sequence at position
18
of the SNR6 gene and to the ensuing inhibition of TFIIIB
assembly (27). Particularly informative was the case of a
tRNAAsn gene having the more complex terminator sequence
T5CT4CT13 (Fig. 5B,
lanes 8-12). In this gene, the first T5 element
induces termination by about half of the elongating polymerases with
the production of a 95-nt-long RNA, whereas pol III molecules reading
through this sequence end up at the more distal T13 site
and produce a 106-nt-long transcript (lane 8). The
T10 PNA can only bind to the latter element with a small
(±3 nt) positional heterogeneity. As shown in Fig. 5B
(lanes 10-12) a PNA bound to such an element not only
causes the disappearance of the T13-terminated transcript but also determines a strong reduction in the levels of the
T5-terminated transcripts, with the appearance of two early
terminated products (bracketed in lane 11) 15-20
nt shorter than the full-length (106 nt) transcript. Considering that 5 U residues, generated by transcription through the first 5 bp of the
terminator element, are incorporated into the full-length transcripts
(26), the observed transcript-shortening values indicate that the
distance between the polymerase active site and the upstream border of
the (PNA)2-DNA adduct ranges from 10 to 20 bp on different
templates (Fig. 5C).

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Fig. 5.
PNA-induced arrest sites on different class
III genes. A, sizing PNA-induced transcript shortening
on the tRNAIle gene. The tRNAIle gene, either
in a supercoiled form (lanes 1-2, circular) or
after digestion with XhoI (cutting 15 bp upstream of the
first T of the terminator) (lanes 3-4, XhoI) was
transcribed for multiple rounds in the absence ( ) or in the presence
(+) of the T10 PNA (400 nM). The migration
position and length of the complete tDNAIle transcript
(150 nt) and of the XhoI runoff product
(130 nt) are indicated on the right.
B, PNA-induced transcript shortening on other class III
genes. Following preincubation with the indicated concentrations of the
T10 PNA, the tDNAIle(TAT) (lanes
1-2), the SNR6 (lanes 3-7), and the
tDNAAsn(GTT) (lanes 8-12) genes were
transcribed for multiple rounds. The migration positions of transcripts
ending at the T5 or T13 termination sites of
the tRNAAsn gene are indicated on the right. The
main shortened transcript of the SNR6 gene is marked by an
asterisk in lane 4. The two shortened transcripts
of the tRNAAsn gene are bracketed in lane
11. C, schematic representation of a PNA-roadblocked
pol III ternary complex. The double-headed arrow indicates
the distance between the position of transcription arrest and the
upstream border of the (PNA)2-DNA adduct; the polymerase
active site is arbitrarily drawn in the
middle.
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DISCUSSION |
This report provides an in-depth account of the mode of action of
a DNA-binding drug specifically targeting the transcriptional terminators of eukaryotic class III genes. Our analysis utilized a
highly purified RNA polymerase III transcription system from S. cerevisiae in which reinitiation, relying on a facilitated pol
III-recycling mechanism, takes place at high efficiency. Central to
this process is a T-rich terminator element, which by an as yet
unidentified mechanism allows a terminating pol III to efficiently reinitiate by bypassing the slow, de novo enzyme recruitment
step (3). Targeting the complementary poly(dA) tract of the class III
gene terminator with an oligothymine PNA resulted in the formation of a
stable (PNA)2-DNA adduct acting as a sequence-specific
roadblock for elongating pol III. We found that a terminator-located
PNA roadblock exerted two main effects on class III gene transcription. The first one is a dramatic reduction in overall transcription efficiency, due to the fact that pol III molecules get stuck during the
first transcription cycle, with the consequent loss of pol III
recycling. The second, somewhat unexpected effect is the blockage of
transcribing pol III well ahead of the PNA roadblock, as revealed by
the fact that the PNA-arrested ternary complex contains an unreleased
RNA that, depending on the template, is 15-25 nt shorter than the
full-length transcript and does not result from pol III-catalyzed hydrolysis of a longer precursor. This suggests that the polymerization site of a transcribing pol III lies 10-20 bp ahead of the leading edge
of the enzyme that first senses the upstream border of the PNA-DNA
adduct (see Fig. 5C). This distance is larger than that observed in previous studies employing either phage T3 and T7 RNA
polymerases (10) or human RNA polymerase II (8). In these studies, in
which artificial templates containing a
dA10·dT10 cassette were transcribed in the
presence of a T10 PNA, elongation became arrested at the
very upstream border of the PNA-binding site. Although the discrepancy
between pol III and phage RNA polymerases can be explained by the much
larger size of the eukaryotic enzyme, the lack of an earlier arrest in
the case of pol II is more difficult to reconcile with our data. The
possibility that TFIIIC, bound to the intragenic B-block, might
cooperate with a terminator-bound PNA in inducing premature pol III
arrest seems to be ruled out by the fact that an early blockage of
transcription also occurred in the case of the SNR6 gene, in
which the B-block is extragenically positioned 120 bp downstream of the
terminator. Also, incomplete transcripts of the same size were produced
in PNA-inhibited transcription reactions carried out in the presence or
absence of TFIIIC on the tRNAIle gene (data not shown
(16)). A more likely explanation of the observed early blockage of
transcription is that, in the context of the sequence surrounding its
termination site, pol III has an extended conformation that is
especially sensitive to the presence of a downstream roadblock. Indeed,
exonuclease III protection studies conducted on RNA polymerase II
arrested at different positions along the template have shown that the
active site to leading edge distance can vary from 7 to 20 bp depending
on the DNA sequence context of individual positions (28). The range of
these distances is close to what we found with PNA-arrested pol III,
and the variation in transcript shortening observed with different
class III genes may similarly reflect sequence-dependent
changes in the pol III active site to leading edge distance.
Noticeably, a DNA occupancy of about 40 bp has been reported for yeast
pol III having transcribed through the first 17 bp of the
SUP4 tRNA gene (29), a space length that is fully compatible
with an active site to leading edge distance of ~20 bp. The effect of
a protein roadblock on RNA polymerase elongation and termination has
previously been studied by placing either the bacterial lac
repressor (30-33) or a cleavage-defective EcoRI
endonuclease (5, 34) at various positions along the transcribed
sequence. The EcoRI roadblock has been shown to efficiently
arrest both elongating and terminating E. coli RNA
polymerases, and a detailed analysis of the arrested complexes has
suggested a model in which the leading edge of the transcribing
polymerase precedes by 7-9 bp the site at which polymerization occurs
(34). More consistent with our observations, studies of bacterial RNA
polymerase blockage by a psoralen diadduct have shown that the leading
edge of the enzyme can extend further downstream, being separated by as
much as 18-20 bp from the growing point of the RNA chain (35).
The T10 PNA very effectively inhibited transcription of
various class III genes with a half-inhibitory concentration (100 nM) 5- to 10-fold lower than that previously determined for
phage (10) and human RNA polymerases (8). This difference is likely due
to the negatively supercoiled state of the templates utilized for our
pol III experiments, because we observed a 10-fold increase of the PNA
half-inhibitory concentration upon linearization of the
tDNAIle-containing plasmid. By influencing the dynamics of
base pair breathing, DNA supercoiling enhances PNA binding (19), a most important effect in view of the possible in vivo use of PNAs
as negative modulators of chromatin-assembled (i.e.
supercoiled) target genes. Because the pol III transcription machinery,
unlike pol I and pol II, efficiently terminates and reinitiates
in vitro on supercoiled templates, it lends itself as the
most sensitive and biochemically appropriate system for studying the
anti-transcriptional activity of PNAs.
The possibility of controlling eukaryotic gene transcription with small
effector molecules has important pharmacological implications. Several
studies have recently demonstrated the transcriptional inhibition of
specific genes in vitro and in vivo by
triple-helix forming oligonucleotides (36, 37), synthetic polyamides
containing N-methylimidazole and N-methylpyrrole
amino acids (38, 39), and PNAs (40-43). In all of these studies, DNA
ligands were designed so as to target specific sequences either
involved in transcription initiation, thereby inhibiting transcription
complex assembly, or located in the middle of a transcriptional unit to
block transcription elongation. Our present findings extend the above
results by showing that targeting a well-conserved control element at
the end of class III transcriptional units also causes an elongation
arrest. Eukaryotic genes transcribed by RNA polymerase III, especially the tRNA and the 5 S rRNA genes, are novel potential targets of anti-gene strategies. In fact, because of their key roles in protein biosynthesis, the products of these genes are essential for cell proliferation, and their deregulation in neoplastic cells has been
proposed to contribute to the loss of cell growth control that
accompanies tumor formation (44). The present demonstration that
oligothymine PNAs strongly and specifically inhibit pol III transcription thus sets the ground for future studies aimed to optimize
the in vivo exploitation of these class III terminator ligands for pharmacological purposes.
 |
ACKNOWLEDGEMENTS |
We are grateful to Christophe Carles and
Emmanuel Favry for the gift of purified RNA polymerase III, and to
Claudio Rivetti for helpful suggestions and comments on the manuscript.
Encouragement and support from Gian Luigi Rossi are also gratefully acknowledged.
 |
FOOTNOTES |
*
This research was supported by grants from the National
Research Council of Italy and from the Ministry of University and Scientific and Technological Research (Rome, Italy; Cofin Project 99).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence may be addressed: Tel.: 39-0521-905646; Fax:
39-0521-905151; E-mail: s.ottonello@unipr.it (for S. O.); gdieci{at}unipr.it (for G. D.).
Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M009367200
 |
ABBREVIATIONS |
The abbreviations used are:
pol, polymerase;
PNA, peptide nucleic acid;
TFA, trifluoroacetic acid;
MIPS, Munich
Information Center for Protein Sequences;
bp, base pair(s);
T10 PNA, H-T10-D-Lys-NH2 PNA;
nt, nucleotide(s).
 |
REFERENCES |
1.
|
Kassavetis, G. A.,
Braun, B. R.,
Nguyen, L. H.,
and Geiduschek, P. E.
(1990)
Cell
60,
235-245[Medline]
[Order article via Infotrieve]
|
2.
|
Kassavetis, G. A.,
Kumar, A.,
Letts, G. A.,
and Geiduschek, E. P.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9196-9201[Abstract/Free Full Text]
|
3.
|
Dieci, G.,
and Sentenac, A.
(1996)
Cell
84,
245-252[Medline]
[Order article via Infotrieve]
|
4.
|
Allison, D. S.,
and Hall, B. D.
(1985)
EMBO J.
4,
2657-2664[Abstract]
|
5.
|
Nudler, E.,
Kashlev, M.,
Nikiforov, V.,
and Goldfarb, A.
(1995)
Cell
81,
351-357[Medline]
[Order article via Infotrieve]
|
6.
|
Nielsen, P. E.
(1999)
Curr. Opin. Biotechnol.
10,
71-75[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Nielsen, P. E.,
Egholm, M.,
and Buchardt, O.
(1994)
J. Mol. Recognit.
7,
165-170[Medline]
[Order article via Infotrieve]
|
8.
|
Hanvey, J. C.,
Peffer, N. J.,
Bisi, J. E.,
Thomson, S. A.,
Cadilla, R.,
Josey, J. A.,
Ricca, D. J.,
Hassman, C. F.,
Bonham, M. A.,
Au, K. G.,
Carter, S. G.,
Bruckenstein, D. A.,
Boyd, A. L.,
Noble, S. A.,
and Babiss, L. E.
(1992)
Science
258,
1481-1485[Medline]
[Order article via Infotrieve]
|
9.
|
Nielsen, P. E.,
Egholm, M.,
Berg, R. H.,
and Buchardt, O.
(1993)
Anticancer Drug Des.
8,
53-63[Medline]
[Order article via Infotrieve]
|
10.
|
Nielsen, P. E.,
Egholm, M.,
and Buchardt, O.
(1994)
Gene
149,
139-145[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Larsen, H. J.,
and Nielsen, P. E.
(1996)
Nucleic Acids Res.
24,
458-463[Abstract/Free Full Text]
|
12.
|
Haaima, G.,
Lohse, A.,
Buchardt, O.,
and Nielsen, P. E.
(1996)
Angew. Chem. Int. Ed. Engl.
35,
1939-1942
|
13.
|
Püschl, A.,
Sforza, S.,
Haaima, G.,
Dahl, O.,
and Nielsen, P. E.
(1998)
Tetrahedron Lett.
39,
4707-4710[CrossRef]
|
14.
|
Christensen, L.,
Fitzpatrick, R.,
Gildea, B.,
Petersen, K. H.,
Hansen, H. F.,
Koch, T.,
Egholm, M.,
Buchardt, O.,
Nielsen, P. E.,
Coull, J.,
and Berg, R. H.
(1995)
J. Pept. Sci.
3,
175-183
|
15.
| Sforza, S., Ghirardi, S., Corradini, R., Dossena, A., and Marchelli, R. (2000) Eur. J. Org. Chem. 2905-2913
|
16.
|
Dieci, G.,
Percudani, R.,
Giuliodori, S.,
Bottarelli, L.,
and Ottonello, S.
(2000)
J. Mol. Biol.
299,
601-613[CrossRef][Medline]
[Order article via Infotrieve]
|
17.
|
Gabrielsen, O. S.,
and Oyen, T. B.
(1987)
Nucleic Acids Res.
15,
5699-5713[Abstract]
|
18.
|
Burnol, A. F.,
Margottin, F.,
Schultz, P.,
Marsolier, M. C.,
Oudet, P.,
and Sentenac, A.
(1993)
J. Mol. Biol.
233,
644-658[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Bentin, T.,
and Nielsen, P. E.
(1996)
Biochemistry
35,
8863-8869[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Dieci, G.,
Hermann-Le Denmat, S.,
Lukhtanov, E.,
Thuriaux, P.,
Werner, M.,
and Sentenac, A.
(1995)
EMBO J.
14,
3766-3776[Abstract]
|
21.
|
Nielsen, P. E.,
Egholm, M.,
Berg, R. H.,
and Buchardt, O.
(1991)
Science
254,
1497-1500[Medline]
[Order article via Infotrieve]
|
22.
| Sforza, S., Haaima, G., Marchelli, R., and Nielsen, P. E. (1999)
Eur. J. Org. Chem. 197-204
|
23.
|
Lagriffoule, P.,
Wittung, P.,
Eriksson, M.,
Jensen, K. K.,
Nordén, B.,
Buchardt, O.,
and Nielsen, P. E.
(1997)
Chem. Eur. J.
3,
912-919
|
24.
|
Whitehall, S. K.,
Bardeleben, C.,
and Kassavetis, G. A.
(1994)
J. Biol. Chem.
269,
2299-2306[Abstract/Free Full Text]
|
25.
|
Bobkova, E. V.,
and Hall, B. D.
(1997)
J. Biol. Chem.
272,
22832-22839[Abstract/Free Full Text]
|
26.
|
Matsuzaki, H.,
Kassavetis, G. A.,
and Geiduschek, E. P.
(1994)
J. Mol. Biol.
235,
1173-1192[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Gerlach, V. L.,
Whitehall, S. K.,
Geiduschek, E. P.,
and Brow, D. A.
(1995)
Mol. Cell. Biol.
15,
1455-1466[Abstract]
|
28.
|
Samkurashvili, I.,
and Luse, D. S.
(1996)
J. Biol. Chem.
271,
23495-23505[Abstract/Free Full Text]
|
29.
|
Bartholomew, B.,
Durkovich, D.,
Kassavetis, G. A.,
and Geiduschek, E. P.
(1993)
Mol. Cell. Biol.
13,
942-952[Abstract]
|
30.
|
Deuschle, U.,
Gentz, R.,
and Bujard, H.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
4134-4137[Abstract]
|
31.
|
Deuschle, U.,
Hipskind, R. A.,
and Bujard, H.
(1990)
Science
248,
480-483[Medline]
[Order article via Infotrieve]
|
32.
|
Sellitti, M. A.,
Pavco, P. A.,
and Steege, D. A.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
3199-3203[Abstract]
|
33.
|
Jeong, S. W.,
Lang, W. H.,
and Reeder, R. H.
(1995)
Mol. Cell. Biol.
15,
5929-5936[Abstract]
|
34.
|
Pavco, P. A.,
and Steege, D. A.
(1990)
J. Biol. Chem.
265,
9960-9969[Abstract/Free Full Text]
|
35.
|
Shi, Y. B.,
Gamper, H.,
Van Houten, B.,
and Hearst, J. E.
(1988)
J. Mol. Biol.
199,
277-293[Medline]
[Order article via Infotrieve]
|
36.
|
Praseuth, D.,
Guieysse, A. L.,
and Hélène, C.
(1999)
Biochim. Biophys. Acta
1489,
181-206[Medline]
[Order article via Infotrieve]
|
37.
|
Faria, M.,
Wood, C. D.,
Perrouault, L.,
Nelson, J. S.,
Winter, A.,
White, M. R.,
Helene, C.,
and Giovannangeli, C.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3862-3867[Abstract/Free Full Text]
|
38.
|
Gottesfeld, J. M.,
Neely, L.,
Trauger, J. W.,
Baird, E. E.,
and Dervan, P. B.
(1997)
Nature
387,
202-205[CrossRef][Medline]
[Order article via Infotrieve]
|
39.
|
Dickinson, L. A.,
Gulizia, R. J.,
Trauger, J. W.,
Baird, E. E.,
Mosier, D. E.,
Gottesfeld, J. M.,
and Dervan, P. B.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
12890-12895[Abstract/Free Full Text]
|
40.
|
Cutrona, G.,
Carpaneto, E. M.,
Ulivi, M.,
Roncella, S.,
Landt, O.,
Ferrarini, M.,
and Boffa, L. C.
(2000)
Nat. Biotechnol.
18,
300-303[CrossRef][Medline]
[Order article via Infotrieve]
|
41.
|
Mologni, L.,
Nielsen, P. E.,
and Gambacorti-Passerini, C.
(1999)
Biochem. Biophys. Res. Commun.
264,
537-543[CrossRef][Medline]
[Order article via Infotrieve]
|
42.
|
Gambacorti-Passerini, C.,
Mologni, L.,
Bertazzoli, C.,
le-Coutre, P.,
Marchesi, E.,
Grignani, F.,
and Nielsen, P. E.
(1996)
Blood
88,
1411-1417[Abstract/Free Full Text]
|
43.
|
Vickers, T. A.,
Griffith, M. C.,
Ramasamy, K.,
Risen, L. M.,
and Freier, S. M.
(1995)
Nucleic Acids Res.
23,
3003-3008[Abstract]
|
44.
|
White, R. J.
(1997)
Trends Biochem. Sci.
22,
77-80[CrossRef][Medline]
[Order article via Infotrieve]
|
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