Selectivity and proofreading both contribute
significantly to the fidelity of RNA polymerase
III transcription
Nazif Alic†, Nayla Ayoub, Emilie Landrieux‡, Emmanuel Favry, Peggy Baudouin-Cornu, Michel Riva§,
and Christophe Carles
Commissariat à l’Énergie Atomique, Institut de Biologie et de Technologies de Saclay, F-91191 Gif sur Yvette Cedex, France
Communicated by E. Peter Geiduschek, University of California at San Diego, La Jolla, CA, May 3, 2007 (received for review December 8, 2006)
We examine here the mechanisms ensuring the fidelity of RNA
synthesis by RNA polymerase III (Pol III). Misincorporation could
only be observed by using variants of Pol III deficient in the intrinsic
RNA cleavage activity. Determination of relative rates of the
reactions producing correct and erroneous transcripts at a specific
position on a tRNA gene, combined with computational methods,
demonstrated that Pol III has a highly efficient proofreading
activity increasing its transcriptional fidelity by a factor of 103 over
the error rate determined solely by selectivity (1.8 ⴛ 10ⴚ4). We
show that Pol III slows down synthesis past a misincorporation to
achieve efficient proofreading. We discuss our findings in the
context of transcriptional fidelity studies performed on RNA Pols,
proposing that the fidelity of transcription is more crucial for Pol III
than Pol II.
nucleotide selectivity 兩 transcriptional fidelity
B oth the replication and expression of genetic material occur
with extremely high fidelity. During genome replication in
Escherichia coli, one error is created per 109 nucleotides incor-
porated (1, 2). This high fidelity results in part from the high
accuracy of DNA synthesis by DNA-dependent DNA poly-
merases (Pols). The mechanisms whereby these enzymes ensure
the fidelity of DNA synthesis are well characterized. DNA Pols
are highly selective for the correct nucleotide, using it 103–106
times more efficiently than an incorrect one. Furthermore, any
errors created can be immediately corrected by an intrinsic
proofreading nuclease activity (2). Importantly, different DNA
Pols may have a different level of fidelity, being optimized for the
particular context in which they perform DNA synthesis (2–4).
The expression of genetic material is also highly accurate. In E.
coli cells under standard growth conditions, the error rate of
transcription is 10⫺5. Theoretical considerations argue in favor of
the importance of transcriptional fidelity for the cell (5). Several
experimental approaches have confirmed this importance in vivo
and especially under conditions favoring nucleotide triphosphate
(NTP) misincorporation (6–8). However, only a few studies have
investigated the molecular mechanisms that underlie the fidelity of
RNA synthesis by DNA-dependent RNA Pols.
Presteady-state kinetic analyses of nucleotide addition by the E.
coli RNA Pol indicated that synthesis occurs through a branched
kinetic mechanism with the enzyme switching between an activated
state and a state termed ‘‘unactivated’’ at each template position.
This unactivated state, absent from DNA Pols, is characterized by
an extremely slow phosphotransfer reaction and can be reversed to
the active state only in the presence of the correct NTP (9, 10).
Fidelity of transcription is ensured by the selectivity of the Pol for
the correct NTP and by kinetic trapping of the ternary complexes
in the unactivated state in the absence of the correct NTP or after
a misincorporation (9). Furthermore, recent studies have demon-
strated that transcriptional fidelity is intimately linked to the
mechanism of elongation, be it through the flexibility of the F
bridge domain (11), the preloading of NTPs and dynamic error
10400 –10405 兩 PNAS 兩 June 19, 2007 兩 vol. 104 兩 no. 25
correction (12), or the association of the trigger loop with the
matched NTP in the A site, thus coupling NTP recognition and
phosphodiester bond catalysis (13).
Initially, unlike DNA Pols, RNA Pols had been thought to
contain no proofreading activity. RNA-cleavage within ternary
complexes was subsequently noted for RNA Pols from all domains
of life (14–17). Unlike DNA Pols, this activity resides in the Pol
active site and for it to be efficient the E. coli RNA Pol requires
accessory proteins, GreA or GreB (18, 19). GreA/B can improve
the overall fidelity of the E. coli RNA Pol (9). Similarly, TFS, the
archeal functional equivalent of GreA/B, reduces the steady-state
error rate of the archaeal RNA Pol (20).
In eukaryotes, three similar but distinct forms of RNA Pol
transcribe each a different set of genes in the nuclear genome. Only
the fidelity of the mRNA-synthesizing Pol II has been investigated
to date. The selectivity of Pol II for the correct NTP was measured
in vitro (21), and, paralleling the situation in E. coli, the role of
TFIIS, the Pol II cleavage-stimulatory factor, in permitting efficient
proofreading was determined (21, 22). Interestingly, at least in the
yeast Saccharomyces cerevisiae, the errors committed by Pol II
during the synthesis of mRNAs may be masked by the higher
error-rate of mRNA translation (23). In E. coli during translation
one error occurs per 104 residues (5).
The two other nuclear Pols, Pol I and Pol III, synthesize only
untranslated RNAs. Thus, the fidelity of their transcription may be
of even greater importance for the cell; however, their fidelity has
not been examined. Elongation complexes of Pol I appear to
contain a dissociable TFIIS-like activity (24). On the other hand,
Pol III appears to be unique, because, unlike Pol I and II, it has a
high intrinsic RNA-cleavage activity that does not require an
accessory factor (16).
In this study, we examined nucleotide misincorporation by
cleavage-competent and cleavage-deficient S. cerevisiae Pol III
ternary complexes at a specific position on a tRNA gene. The
results define the strategies used by a eukaryotic RNA Pol with high
intrinsic cleavage activity to ensure the accuracy of its transcription.
We used computational methods to combine the experimental
observations and predict the steady-state error rate of Pol III and
Author contributions: N. Alic, E.L., M.R., and C.C. designed research; N. Alic, N. Ayoub, E.L.,
E.F., and M.R. performed research; E.L. contributed new reagents/analytic tools; P.B.-C.
performed computation analyses and simulations; N. Alic, E.L., P.B.-C., M.R., and C.C.
analyzed data; and N. Alic, P.B.-C., M.R., and C.C. wrote the paper.
The authors declare no conflict of interest.
Abbreviations: Pol, polymerase; NTP, nucleotide triphosphate.
†Present address: Department of Biology, University College London, Gower Street, London
WC1E 6BT, United Kingdom.
‡Present address: CMU/Département de Microbiologie et de Médecine Moléculaire,
Rue Michel Servet 1, 11211 Geneva, Switzerland.
§To whom correspondence should be addressed. E-mail: michel.riva@cea.fr.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0704116104/DC1.
© 2007 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0704116104
Fig. 1. A Pol III form devoid of RNA cleavage activity incorporates a mispaired
nucleotide. (A) Template-strand sequence in the beginning of SUP4 (gray) and
the sequence of the 17-mer RNA (black). (B) (Upper) Shows the relevant
sequence of SUP4 (gray) and the 3⬘-terminal sequence of the 17-mer (black),
with X denoting the position at which the NTP incorporation was tested.
(Lower) Purified ternary complexes containing radiolabeled 17-mer SUP4, Pol
III, or Pol III⌬ were analyzed either directly (lanes 1 and 6) or after a 5-min
incubation with 600 ␮M of the noted NTP. Arrow, residual 18-mer; asterisks,
slippage product. (C) SUP4 was transcribed for 5 min in the presence of ATP,
CTP and radiolabeled UTP by Pol III, Pol III⌬ alone, or Pol III⌬ preincubated for
10 min with the indicated purified recombinant subunits.
to demonstrate the equally high contribution of proofreading and
nucleotide selectivity to the overall fidelity of Pol III.
Results
Pol III⌬, an Incomplete Form of Pol III Lacking RNA-Cleavage Activity,
Can Quantitatively Incorporate an Incorrect Nucleotide. To gain an
insight into the mechanisms ensuring the fidelity of Pol III tran-
scription, we initially examined the ability of the wild-type Pol III
to incorporate an incorrect base. Transcription by Pol III of the
SUP4 template preassembled with transcription factors TFIIIC and
TFIIIB was initiated by the addition of ATP, CTP, and radiolabeled
UTP. Pol III formed stable ternary complexes containing a 17-mer
RNA, due to the absence of GTP required for synthesis at positions
⫹18 and ⫹19 of the template (Fig. 1 A and B). These were purified
from free NTPs and further incubated with each of the four NTPs.
As expected, the 17-mer was correctly extended to a 19-mer in the
presence of GTP (Fig. 1B, lane 2) whereas no synthesis was
observed with the other NTPs (Fig. 1B, lanes 3–5). Instead, the
intrinsic RNA cleavage activity of Pol III led to the disappearance
of the 17-mer generating shorter RNAs (Fig. 1B, lanes 3–5). In
contrast to this apparent inability of Pol III to incorporate a
mispaired nucleotide, Chédin et al. (25) have reported that tran-
scription of SUP4 in the absence of GTP by Pol III⌬, an incomplete
form of Pol III lacking the C11, C37 and C53 subunits, resulted in
formation of an 18-mer transcript differing at the 3⬘ end from the
17-mer produced by Pol III (25, 26). The authors suggested that Pol
III⌬ incorporated a mispaired nucleotide at position ⫹18, which
failed to be subsequently removed because of the lack of RNA-
cleavage activity in Pol III⌬ resulting from the absence of the
TFIIS-like C11 subunit (25). We wanted to test this hypothesis and
determine which base was misincorporated at position ⫹18. The
SUP4 gene was transcribed in the absence of GTP by Pol III⌬
preincubated with the recombinant C11 subunit (rC11). The re-
sulting halted ternary complexes contained a 17-mer RNA and
were purified to remove rC11 and the NTPs (25, 26). Traces of the
18-mer transcript remained, which could be attributed to a some-
what reduced cleavage efficiency of rC11 compared with the
endogenous C11, and a negligible amount of a slippage product (27)
was observed (Fig. 1B, lane 6). The purified complexes were assayed
for their capacity to incorporate a single nucleotide at position ⫹18.
In the presence of ATP, Pol III⌬ quantitatively synthesized an
18-mer (Fig. 1B, lane 8). This 18-mer could not have resulted from
incorporation of ITP that might have contaminated our ATP
Alic et al.
BIOCHEMISTRY
Fig. 2. Pol III selectivity determined in the absence of RNA-cleavage activity.
(A) (Upper) Shows the relevant sequence of SUP4 (gray) and the 3⬘-terminal
sequence of the 17-mer (black) with the newly added bases in bold. (Lower)
Time-course analysis of incorporation of GTP (0.6 ␮M) by ternary complexes
containing SUP4, Pol III⌬ and the radiolabeled 17-mer. For each reaction time,
the amount of the residual 17-mer was quantified and expressed as percent-
age of the initial amount (t ⫽ 0) after subtraction of the lowest quantity of
17-mer remaining unreacted. Data were fitted to a single-exponential func-
tion and the apparent rate constant (ka) calculated. (B) Same as in A, except
that the ternary complexes were incubated with ATP (600 ␮M) instead of GTP.
The mismatch formed is framed on the sequence representation. (C) Same as
in A except that the ternary complexes were preincubated for 10 min with the
cleavage-incompetent rC11E92H and r(C37-C53), before being allowed to react
with either GTP (0.6 ␮M) (Left) or ATP (600 ␮M) (Right).
stocks, because in the presence of ITP, Pol III⌬ produces a 19-mer
(data not shown) and a 19-mer was not observed after 5 min (Fig.
2B) nor after 60 min of incubation with ATP (data not shown).
Thus, the appearance of an 18-mer revealed that the enzyme
incorrectly incorporates an A instead of a G. Misincorporation of
UTP occurred less efficiently (Fig. 1B, lane 10), whereas no
incorporation of CTP was observed (Fig. 1B, lane 9). As for the
wild-type enzyme, a 19-mer transcript was synthesized in the
presence of GTP (Fig. 1B, lane 7). However, on longer incubation
in the presence of GTP, Pol III⌬ also synthesized a 20-mer
transcript, indicating that it misincorporated a G instead of a U at
this position (data not shown). The results demonstrated that Pol
III⌬ is capable of misincorporation.
RNA Cleavage Activity of Pol III Is Sufficient to Prevent Observable
Misincorporations. From the above observations, we could not
conclude whether Pol III⌬ produced quantitatively a mispaired
transcript solely because it lacked RNA-cleavage activity or because
the missing C11, C37, and/or C53 subunits were also important in
preventing misincorporation. Therefore, it remained unclear
whether Pol III did not incorporate an incorrect base to an
observable degree, or whether misincorporation occurred but was
masked by the efficient subsequent cleavage of the RNA. To
address this issue, we used a Pol III form containing a complete set
PNAS 兩 June 19, 2007 兩 vol. 104 兩 no. 25 兩 10401
of subunits but lacking the intrinsic RNA-cleavage activity. We have
recently shown that Pol III⌬ reconstituted with rC37, rC53, and the
mutant rC11E92H subunit displays the same transcriptional prop-
erties in all of the in vitro tests examined (initiation, elongation rate,
termination, and reinitiation efficiency) as the wild-type enzyme
except that it lacks RNA-cleavage activity (26). When transcription
of SUP4 was carried out in the absence of GTP, Pol III⌬ alone
synthesized an 18-mer (Fig. 1C, lane 2), whereas Pol III⌬ preincu-
bated with the three wild-type recombinant subunits, and the
wild-type enzyme synthesized a 17-mer RNA (Fig. 1C, lanes 3 and
1, respectively). However, when we used the rC11E92H mutant
subunit instead of the wild-type rC11, an 18-mer was synthesized
(Fig. 1C, lane 4). These observations strongly suggest that the
synthesis of a 17-mer by the wild-type Pol III reflects a steady state
in which misincorporations at position ⫹18 occur but are rapidly
corrected by the efficient RNA cleavage activity intrinsic to Pol III.
Pol III Incorporates a Correct Base 103 Times More Efficiently Than an
Incorrect One. The errors committed by Pol III⌬ allowed us to
characterize the specificity of nucleotide incorporation by Pol III.
We determined the incorporation rates for the correct and an
incorrect nucleotide at position ⫹18 of the SUP4 gene in a
time-course analysis of single nucleotide addition to a 17-mer within
Pol III⌬ ternary complexes. The incorporation of the correct
nucleotide (GTP) proceeding extremely rapidly, the reaction was
slowed by reducing the GTP concentration to 0.6 ␮M. Under these
conditions, the reaction was virtually complete by 15 s (Fig. 2 A).
Because the quantity of GTP greatly exceeds that of the ternary
complexes in the reaction, the concentration of GTP can be
considered to remain constant and the incorporation of G to occur
with pseudo-first-order kinetics. By fitting a single exponential
equation to the time-dependent loss of the 17-mer, the pseudo-rate
constant (ka) for the incorporation of the correct nucleotide (GTP)
was estimated to be 0.22 s⫺1 with a standard error of 0.04 s⫺1 (Fig.
2 A). The incorporation of the preferred incorrect nucleotide
(ATP) was much slower, taking 2.5 min for the reaction to go to
completion in the presence of 600 ␮M ATP (Fig. 2B). The
pseudo-rate constant for this reaction was 0.039 ⫾ 0.004 s⫺1 (Fig.
2B). When the concentration of ATP was reduced to 60 ␮M, the
pseudo-rate constant was equivalently reduced by an order of
magnitude [supporting information (SI) Fig. 6], indicating that
ATP was not saturating at 600 ␮M, and allowing us to extrapolate
the rate of ATP incorporation at 0.6 ␮M to 3.9 ⫻ 10⫺5 s⫺1. Hence,
the correct base was incorporated ⬇6 ⫻ 103 times faster than the
preferred incorrect one at nucleotide concentrations below the
effective Km for both the correct and incorrect nucleotide.
Because our measurements were performed with Pol III⌬, it was
important to determine whether the selectivity observed reflected
that of the wild-type Pol III deprived only of RNA cleavage activity.
We used the rC11E92H mutant subunit, together with the r(C37-
C53) complex, to reconstitute a Pol III lacking only its intrinsic
RNA cleavage activity (26). Examination of the misincorporation
rate for this enzyme form revealed it to have the same rate of
addition of the correct and incorrect nucleotide as Pol III⌬ (Fig.
2C). Thus, the selectivity measured for Pol III⌬ also reflects the
selectivity of the wild-type Pol III.
RNAs Containing a 3ⴕ-End Mismatch Are Cleaved More Rapidly Than
Those Containing a Correctly Paired End. To clarify the importance
of the RNA cleavage activity versus selectivity in ensuring the
fidelity of Pol III transcription, we sought to quantify the rate of the
former reaction. Pol III ternary complexes containing a 17-mer
were purified and allowed to cleave the RNA in transcription buffer
in the absence of NTPs (Fig. 3A). We determined the apparent rate
constant for the cleavage of an RNA with a correctly paired 3⬘ end
from the time-dependent loss of the 17-mer (Fig. 3A) to be 0.10 ⫾
0.01 s⫺1. However, the important parameter in proofreading by Pol
III is the rate of cleavage of an RNA containing a mispaired
10402 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0704116104
Fig. 3. Time-course analysis of RNA cleavage by Pol III. (A) Time-course of
RNA cleavage in transcription buffer in the absence of NTPs by purified ternary
complexes containing SUP4, Pol III, and the radiolabeled 17-mer. Data were
analyzed as described for Fig. 2 A. (B) Purified ternary complexes containing
SUP4, PolIII⌬ and either the (radiolabeled) correctly 3⬘-end-paired 17-mer or
3⬘-end mispaired 18-mer transcript were preincubated for 10 min with wild-
type rC11 and r(C37–C53) and the RNA-cleavage reaction was initiated by
starting incubation in cleavage buffer (at 16°C). The data were analyzed as
described for Fig. 2 A.
nucleotide at its 3⬘-end. This rate cannot be directly measured,
because the mispaired 18-mer is undetectable and supposedly
short-lived within Pol III ternary complexes. Therefore, purified
ternary complexes containing Pol III⌬ and the 3⬘ end-mispaired
18-mer were incubated with rC11 and r(C37–C53). We compared
the rate of cleavage of the 18-mer in such reconstituted wild-type-
like ternary complexes to the rate of cleavage of the correct 17-mer
transcript in similarly obtained complexes. Because the cleavage of
the 18-mer was too rapid at 25°C, the reactions were performed in
cleavage buffer at 16°C. Despite the reconstituted Pols cleaving only
up to 40% of the RNA initially present (SI Fig. 7), we clearly
observed that the 18-mer transcript was cleaved 8 times more
rapidly than the 17-mer (Fig. 3B). This higher rate of the 18-mer
cleavage was not due to a different sequence context (position 18
versus 17), because an 18-mer transcript ending with a 3⬘-OMeG
was cleaved at a rate very similar to that of the 17-mer (SI Fig. 8).
Thus, the cleavage of a 3⬘-end mispaired transcript by Pol III is more
efficient than the cleavage of a correctly paired one, indicating that
the enzyme discriminates between correct and incorrect bases
during both synthesis and cleavage reactions. We extrapolated from
the rate of 18-mer cleavage relative to that of the 17-mer by the
reconstituted Pol III to the wild-type Pol III, estimating the rate of
cleavage of the 3⬘-end mispaired 18-mer transcript at 25°C by Pol
III to be 0.83 s⫺1 (for details, see ‘‘Note 1’’ in SI Text).
Misincorporation Slows the Incorporation of the Next Nucleotide. To
appreciate the importance of the cleavage reaction in the fidelity of
Pol III, we had to determine the rate of the competing step, the
addition of the correct nucleotide after a misincorporation. In the
presence of 600 ␮M GTP, purified Pol III⌬ ternary complexes
extended the 3⬘-end mispaired 18-mer to a 19-mer that subse-
quently disappeared giving a 20-mer transcript (Fig. 4). Essentially
all of the initially present 18-mer was lost by 60 min (Fig. 4). The
simplest explanation for the transient appearance of a 19-mer
followed by formation of a 20-mer is that the 20-mer arises from a
misincorporation of GTP instead of UTP at position ⫹20 and that
the rate-limiting step in the formation of the 20-mer is the passage
from ⫹18 to ⫹19, i.e., the incorporation of a correct base after a
mispaired base. The apparent rate constant for disappearance of
the 18-mer was 94 ⫻ 10⫺5 ⫾ 4 ⫻ 10⫺5 s⫺1 (Fig. 4). Thus, the
incorporation of a correct base after a mispaired base is ⬇105 times
slower than after a correctly paired one. Furthermore, our analyses
demonstrate that the addition of a ribonucleotide after a misincor-
poration is the slowest step in the synthesis of error-containing
Alic et al.
 Table 1. Summary of the rate constants determined in this study
Complex* ka (s⫺1) Ref. in Fig. 5

Incorporation† 5⬘ѧCG 2.2 10⫺1‡ k1

3⬘ѧGCCѧ5⬘
5⬘ѧCA 3.9 ⫻ 10⫺2 k2
3⬘ѧGCCѧ5⬘
5⬘ѧCAG 9.4 ⫻ 10⫺4 k3
3⬘ѧGCCѧ5⬘
Cleavage 5⬘ѧC 1 ⫻ 10⫺1 k4
Fig. 4. Addition of a correct nucleotide after misincorporation is extremely
3⬘ѧGCCѧ5⬘
inefficient. (A) (Upper) The relevant sequence of SUP4 (gray) and the 3⬘-
terminal sequence of the 18-mer (black) with the newly added bases in bold 5⬘ѧCA 8.3 ⫻ 10⫺1§ k5
and the mismatches framed. (Lower) The time-course analysis of GTP (600 ␮M) 3⬘ѧGCCѧ5⬘
incorporation by purified ternary complexes containing SUP4, Pol III⌬, and the
*The upper sequence shows the 3⬘-end of the RNA, the lower sequence shows
radiolabeled 3⬘-end mispaired 18-mer transcript. (B) The data were analyzed
the template DNA strand, and mispaired RNA bases are italicized.
as described for Fig. 2 A. †The incorporation of the underlined base is shown. The corresponding NTP is

at 600 ␮M unless noted.

‡The measurement was made at 0.6 ␮M GTP.
transcripts by Pol III. In contrast, this step is not slower than the §Determined from the relative rate of cleavage of 18-mer vs. 17-mer and the
actual misincorporation in the case of E. coli RNA Pol and Pol II measured rate constant for the 17-mer cleavage (see Note 1 in SI Text).
(9, 21).

Selectivity and Proofreading Contribute Equally to the Fidelity of Pol

III Transcription. We sought a computational approach to integrate
position in an RNA chain is the ratio of the concentrations of the
correct to the incorrect product [n ⫹ 1]/[n* ⫹ 1], which is the inverse

BIOCHEMISTRY
our experimental results and estimate the contributions of proof-
reading and selectivity to the fidelity of Pol III transcription at a of the error rate. We can calculate that, after an infinite time, the
particular position of an RNA chain. Our observations were [n ⫹ 1]/[n* ⫹ 1] ratio reaches:

冉 冊 冉 冊
consistent with the existence of two competing pathways leading to
关n ⫹ 1兴 k 1k 6 k5
an insertion followed by a fixation of either a correct or an incorrect ⫽ 1⫹ [1]
base (Fig. 5). The system of reactions we propose to be important 关n* ⫹ 1兴 t3⬁
k2共k6 ⫹ k4兲 k3
in determining the fidelity of Pol III is characterized by six kinetic
parameters, the first-order rate constants k1–k6 represented in Fig. Assuming that the incorporations of correct ribonucleotides
5. A ternary complex containing a transcript of n ⫺ 1 nucleotides onto a correctly paired 3⬘-end will occur with similar rates
can incorporate at the nth position either a correct nucleotide relative to the rate of misincorporation or extension past a
[giving the product (n) with the first-order rate constant k1] or an misincorporation, we can state that k1 ⫽ k6, giving:

冉 冊 冉 冊
incorrect nucleotide [product (n*), k2]. Each product can either
undergo the cleavage reaction (k4 and k5, respectively), regenerat- 关n ⫹ 1兴 k 12 k5
ing an RNA of n ⫺ 1 nucleotides, or be extended by the incorpo- ⫽ 1⫹ [2]
关n* ⫹ 1兴 t3⬁
k2共k1 ⫹ k4兲 k3
ration of the correct nucleotide to give the (n ⫹ 1) or (n* ⫹ 1)
species, respectively (k6 and k3) (Fig. 5). In essence, the fidelity of Pol III transcription is insured by two
Two simplifications were made in this model: (i) The cleavage of processes: selectivity and proofreading. Selectivity is given by the
the (n) or (n*) species will give a product of length n ⫺ x, with x ⱖ ratio of the rates of incorporation of correct to incorrect
1, but because the extension from position n ⫺ x to n ⫺ 1 (for x ⬎ nucleotide (k1/k2). Proofreading is represented by the ratio of the
1) is rapid, we assumed that the cleavage of the (n) or (n*) RNAs
rate of cleavage of an incorrectly paired nucleotide relative to the
would directly generate the (n ⫺ 1) species. (ii) We did not include
rate of its extension (k5/k3). Indeed, visual inspection of formula
the cleavage of the (n ⫹ 1) or (n* ⫹ 1) transcripts in the model
(Eq. 2) and computer simulations (SI Fig. 9), confirmed that it
because, once a correct nucleotide is incorporated at position n ⫹
is not the absolute rate of cleavage that determines fidelity of Pol
1, the forward synthesis rates may be overwhelming and thus no
longer have a bearing on the misincorporation at position n. We III transcription but the k5/k3 ratio.
discuss later a more complex situation created when this second It is obvious from formula (Eq. 2) that if k1 ⬎⬎ k4 and k5/k3 ⬎⬎
simplification is altered (see Discussion). 1, as is the case observed for Pol III (Table 1), fidelity is given by

冉 冊
One measure of transcriptional fidelity of Pol III at a particular
关n ⫹ 1兴 k1 k5
⬇ ⫻ , [3]
关n* ⫹ 1兴 t3⬁
k2 k3

i.e., it is the product of ‘‘selectivity’’ by ‘‘proofreading’’. The rate

Fig. 5. Modeling the reactions determining Pol III fidelity. Diagram describ-
ing the reactions determining the fidelity of Pol III transcription, where: (n ⫺
1) ⫺ RNA species of length (n ⫺ 1) within a Pol III ternary complex, (n) ⫺
product of the correct incorporation at the template position n, (n*) ⫺ product
of misincorporation at position n, (n ⫹ 1) ⫺ product of the extension of the
correctly paired species (n), and (n* ⫹ 1) ⫺ product of the extension of the
incorrectly paired species (n*).
constants measured in this study for the incorporation of GTP
and misincorporation of ATP at position ⫹18 of SUP4 indicate
that k1/k2 ⫽ 5.6 ⫻ 103 at GTP and ATP concentrations below the
effective Km of the enzyme for these nucleotides. Measurements
of cleavage and extension past the misincorporation indicate that
k5/k3 ⫽ 0.9 ⫻ 103, at NTP ⫽ 600 ␮M. A reduction in [NTP] would
result in lower k3 and higher k5/k3 (see SI Fig. 9). Hence, at
nonsaturating concentrations of nucleotides,
冉 关n ⫹ 1兴
关n* ⫹ 1兴
冊 t3⬁
⫽ 5.0 ⫻ 106, [4]

Alic et al. PNAS 兩 June 19, 2007 兩 vol. 104 兩 no. 25 兩 10403
meaning that Pol III would incorporate one error per 5 ⫻ 106
bases synthesized. Computer simulations revealed that the limit
conditions are reached rapidly (SI Fig. 9).
Therefore, the efficiency of proofreading by Pol III appears to be
within the order of magnitude of the selectivity ratio, indicating that
both processes have a significant contribution to Pol III fidelity.
Discussion
In this study, we show that proofreading is a major determinant
of the fidelity of Pol III transcription. The measurements of
relative reaction rates combined with computer analyses dem-
onstrate that nucleotide selectivity and proofreading contribute
equally to Pol III fidelity at the examined position on the SUP4
gene. Further studies may reveal how these contributions may
vary in different sequence contexts or even in vivo and whether
our conclusions are generally applicable.
Mechanics of Error Avoidance and Error Correction by Pol III. As noted
in the Introduction, few studies have examined the fidelity of RNA
Pols. Still, we find it important to make some comparisons between
the functioning of different RNA Pols, although the comparisons
remain speculative, because they are based on a limited number of
ternary complexes.
The selectivity measured for Pol III (5.6 ⫻ 103) is similar to that
reported for the E. coli RNA Pol and Pol II (103 and ⬎5 ⫻ 102,
respectively) (9, 21), indicating that the selectivity of RNA Pols may
be in the lower end of the range (103–106) reported for DNA Pols
(2). We have measured the rate of the most-likely misincorporation,
which may have led to an underestimate of selectivity. At the same
time, maximal error rates may have also been measured for other
RNA Pols, because preloading of NTPs may reduce their error
incorporation (12).
The proportion of E. coli RNA Pol or Pol II ternary complexes
able to incorporate a correct nucleotide was higher than that
capable of misincorporation (9, 21), which Erie and coworkers
interpreted as indicating that the polymerase could assume an
unproductive conformation (i.e., become ‘‘unactivated’’) at each
synthesis step, thus preventing misincorporation. Single-molecule
analyses on bacterial enzyme confirmed recently this hypothesis
(28). The existence of such an unactivated state was also inferred
for Pol III from detailed analyses of RNA chain elongation (29). We
did not evidence, however, any difference in the proportion of
ternary complexes incorporating the correct or an incorrect nucle-
otide. This observation reflects that the temporal resolution of our
experiments may not be fine enough to allow detection of such a
state. Alternatively, although highly speculative, one cannot for-
mally exclude that Pol III requires intrinsic cleavage activity to enter
an unactivated state, because unlike our study, that of Matsuzaki
and coworkers used a cleavage-competent enzyme (29).
Pol III appears to have an exceptionally high contribution of
proofreading to its fidelity; the magnitude of this contribution, as
determined in this study (103), is high even when compared with the
range reported for the proofreading activities of DNA Pols (few- to
⬎100-fold) (2). Interestingly, both DNA Pols and Pol III rely
strongly on kinetic partitioning between cleavage and extension
reactions for efficient proofreading despite crucial differences in
the mechanism and site of the cleavage activity.
We demonstrated that the rate of cleavage of nascent transcripts
was stimulated by a mismatched base at the 3⬘ end of the RNA. This
may be the extreme case of the preference to cut the nascent RNA
just 5⬘ of an unstable RNA-DNA base pair, demonstrated for Pol
III (30). This extends observations made on RNA-DNA ‘‘dumb-
bell’’ templates, using Pol II⫹TFIIS (22). Importantly, in our study,
the rates of both cleavage and extension are measured under
physiologically more relevant conditions, within authentic ternary
complexes halted on a class III gene.
C11, the protein essential for the cleavage and thus the proof-
reading activity of the polymerase, is a Pol III subunit, making it
10404 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0704116104
very likely that the role of proofreading in the fidelity of the Pol will
be important in different sequence contexts and in vivo. Further-
more, our calculations may have underestimated the contribution of
cleavage to Pol III fidelity. In the model presented (Fig. 5), the
assumption was made that the rate of extension of the (n* ⫹ 1)
species by a correct base is equal to the rate of extension of (n) to
(n ⫹ 1) and cancels out the effects of (n* ⫹ 1) cleavage. However,
precedents exist in DNA Pols for a reduction in the synthesis rates
several bases past the misincorporation, occurring because of
distortions in the active site (31). If we include the extension/
cleavage of the (n* ⫹ 1) species in our model, we find that the
fidelity of transcription, as measured in this case by the (n ⫹ 2):1/m
(n ⫹ 2)/(n* ⫹ 2). (n* ⫹ 2) ratio, increases substantially if the rate
of conversion of (n* ⫹ 1) to (n* ⫹ 2) drops below ⬇2 s⫺1 to reach
4.4 ⫻ 109 when this rate is as low as that of (n*) 3 (n* ⫹ 1) (SI Fig.
10 and SI Text, ‘‘Note 2’’). This increase in fidelity occurs through
an increase in proofreading. The fidelity of Pol III probably lies
between these two extremes: the rate of (n* ⫹ 1) 3 (n* ⫹ 2) is not
as low as that of (n*) 3 (n* ⫹ 1), because on addition of all four
NTPs, the 3⬘-end mispaired 18-mer can be extended to the com-
plete SUP4 transcript in a time-course similar to that in Fig. 4
without the appearance of a 19-mer (data not shown). A further
determinant of the in vivo fidelity is the local NTP concentration,
and computer simulations show that fidelity is linearely propor-
tional to the synthesis rates over at least two orders of magnitude
(data not shown).
RNA Cleavage Activity and in Vivo Properties of Pol III. Even if all
RNA Pols use generally similar mechanisms to achieve high fidelity,
Pol III appears to display specific properties. Why, for example,
does Pol III have a high intrinsic cleavage activity, whereas other
nuclear RNA Pols do not? This property may be due to the
differences in their mechanism of transcription, and reflect their
respective biological roles. With respect to the mechanism of
transcription, the simplest explanation would be that Pol III is less
apt at avoiding transcriptional errors than Pol II. However, the
selectivity of the two polymerases appears comparable.
Currently, the role of the Pol II cleavage factor, TFIIS, in
transcriptional fidelity is unclear. It is not a constitutive partner
of Pol II (32, 33), and there is no evidence for in vivo TFIIS-
mediated Pol II proofreading under standard growth conditions,
i.e., in absence of stress (34). However, conditions favoring
transcriptional errors stimulate the association of TFIIS with
elongating Pol II (33), and the deletion the TFIIS-encoding gene
in yeast (PPR2) causes sensitivity to the same conditions (35).
TFIIS may also have other functions, such as in initiation of
transcription (36) or in elongation (37).
The in vivo nonessential or essential character of the cleavage
activity is another striking difference between the Pol II and the Pol
III systems. PPR2 is not essential for yeast viability (38). Rpb9
subunit of Pol II, a C11 paralogue recently identified as important
for the in vivo fidelity of Pol II and suggested to act in proofreading
(34), is also encoded by a nonessential gene. These findings strongly
suggest that Pol II RNA cleavage activity is not essential in vivo. In
stark contrast, the point mutations in the C11 subunit that abolish
RNA cleavage activity are lethal [double D91A E92A mutant (25);
or E92H mutant, data not shown]. Because the only known
transcriptional defect of Pol III-containing rC11E92H in vitro is the
absence of cleavage activity (26), this activity may be essential for
Pol III in vivo. A simple explanation for this difference would be that
transcriptional fidelity is more crucial for Pol III than for Pol II,
because the former’s untranslated RNA products are direct players
in crucial cellular processes, notably translation. The idea that
eukaryotic RNA Pols may have different accuracies depending on
the class of transcripts they produce was also proposed based on
observations made in E. coli (39). Indeed, with respect to protein-
coding genes, it appears that translation rather than Pol II tran-
Alic et al.
scription is the error-prone process (23), although this may depend
on the gene examined (40).
The essential character of Pol III cleavage activity may also
reflect more profound differences in the in vivo mechanics of Pol
III and II transcription. The efficient transcription of class III genes
proceeds through facilitated reinitiation by the enzyme during
which Pol III has been suggested to remain on the template (41, 42).
The movement of the enzyme on a gene would thus be extremely
reduced, making it difficult, if at all possible, for the cell to
distinguish between truly elongating Pol III and blocked Pols, thus
precluding the possibility of the latter’s degradation to save the
transcription of the gene. In contrast to Pol II, which has been
observed to become ubiquitylated and degraded under the condi-
tions favoring elongation blocs in vivo (43), RNA cleavage and
resynthesis may be the only means for the cell to deal with
elongation-blocked Pol III.
Materials and Methods
Purification of RNA Pol III, Class III Transcription Factors and Expres-
sion and Purification of Recombinant Pol III Subunits. Purification of
Pol III and class III transcription factors was described in ref. 44. Pol
IIIwt was purified from the yMR4 strain, whereas Pol III⌬ was
purified from either C37HA-Ct or yEL7 strain (26). Regardless of
the source, Pol III⌬ behaved the same in all of the tests performed.
Purified recombinant (His7-C37–C53) complex, C11 and C11E92H
were obtained as described (25, 26). rC11, rC11E92H, and the
r(C37–C53) complex were purified to homogeneity (26), as mon-
itored by Coomassie blue staining.
Promoter-Directed in Vitro Transcription, Ternary Complex Purifica-
tion, Single-Nucleotide Addition and Cleavage Assays. NTPs and
[␣-32P]UTP were from Amersham Biosciences (Little Chalfont,
U.K.). Promoter-directed in vitro transcription reactions were per-
formed in 50 ␮l of transcription buffer (20 mM Hepes KOH, pH
7.5/0.1 mM EDTA/5 mM MgCl2/5 mM DTT/5% glycerol/90 mM
KCl). pRS316-SUP4 plasmid (250 ng) was incubated in this buffer
with 50 ng of each rTFIIIC, rTBP, rTFIIIB70, and 0.4 ␮g of B⬙
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reconstituted Pol III (50–100 ng), and a further 20-min incubation.
Transcription was started by addition of 600 ␮M ATP and CTP and
3 ␮M [␣-32P]UTP (400 Ci/mmol) and allowed to proceed for 5 min.
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nucleotide addition and cleavage reactions were performed on
20-␮l aliquots of purified ternary complexes and initiated by the
addition of 20 ␮l of the reaction mix to give the final concentration
of 1⫻ transcription or cleavage buffer with the noted concentration
of an NTP. The reactions were stopped and processed as described
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performed with Image Quant software, Version 1.2. The lowest
quantity of the 17-mer or 18-mer observed in the reactions was
subtracted from all of the values and the data obtained were
BIOCHEMISTRY
normalized so that the amount of the oligomer at time 0 is 100%.
The normalized values were fitted to a single exponential equation
(y ⫽ Ae⫺kx, where A is the initial quantity of oligomer), and the rate
constant (k) was calculated by using Prism 4 software (GraphPad,
San Diego, CA). R2 values were generally ⬎0.98 (all were ⬎0.94).
Computational Analyses. Computational analyses were performed
with MatLab software (MathWorks, Natick, MA). For details, see
SI Text.
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PNAS 兩 June 19, 2007 兩 vol. 104 兩 no. 25 兩 10405