Recognition Sites of 3'-OH Group by T7 RNA Polymerase and Its
Application to Transcriptional Sequencing*
Masaki
Izawa
§,
Nobuya
Sasaki
¶,
Masanori
Watahiki§,
Eiji
Ohara§,
Yuko
Yoneda§,
Masami
Muramatsu
¶,
Yasushi
Okazaki
, and
Yoshihide
Hayashizaki
¶
From the
Genome Science Laboratory, Tsukuba Life
Science Center, The Institute of Physical and Chemical Research
(RIKEN), Koyadai 3-1-1, Tsukuba-city, Ibaraki 305, § Research and Development, Nippon Gene Co., Ltd., 1-29,
Tonya-machi, Toyama 930, and ¶ CREST, Japan Science and Technology
Corp. (JST), Kawaguchi-Center building, 4-1-8 Hon-cho, Kawaguchi,
Saitama 332, Japan
 |
ABSTRACT |
When analyzing the elongation mechanisms in T7
RNA polymerase (T7 RNAP)by using site-directed mutagenesis and a
protein expression system, we identified the recognition sites of the
rNTP 3'-OH group in T7 RNAP. On the basis of three-dimensional crystal
structure analysis, we selected and analyzed six candidate sites
interacting with the 3'-OH group of rNTP in T7 RNAP. We found that the
Phe-644 and Phe-667 sites are responsible for the high selectivity of T7 RNAP for rNTPs. Also, we constructed the protein mutations of these
residues, F644Y and F667Y, which display a >200-fold higher affinity
than the wild type for 3'-dNTPs. These findings indicate that the
phenylalanine residues of 644 and 667 specifically interact with the
3'-OH group. Thus, these mutants, F644Y and F667Y, with incorporation
of 3'-dNTP terminators, which is similar to native rNTPs, can offer low
backgrounds and equal intensities of the sequencing ladders in our
method, called "transcriptional sequencing."
 |
INTRODUCTION |
T7 RNAP1 is a single
99-kDa polypeptide containing in its catalytic domain a highly specific
promoter recognition site and a nascent RNA binding domain. T7 RNAP
displays a stringent specificity for T7 promoter to initiate
transcription. In T7 phage infection and the lytic cycle, T7 RNAP
transcribes class II and III of the T7 phage genome (1). Based on
three-dimensional structure analysis and mutagenesis of the Klenow
fragment, the protein critical regions and structural motifs for
polymerase activity have been suggested. Additionally, from sequence
homologies of the Klenow fragment, Taq DNA polymerase, and
T7 DNA polymerase, structural similarities have also been suggested in
motifs A (532-555 aa), B (625-652 aa), and C (805-818 aa) (2). From
mutagenesis analysis, the critical sites of primer recognition, metal
ion binding, rNTP binding, and polymerase activity have been
determined; however these do not necessarily coincide with the
homologous motifs. Phenylalanine and tyrosine have been reported to
play important roles in discriminating 2'- and 3'-OH groups in
Escherichia coli DNA polymerase I (3, 4) and Taq
DNA polymerase (5). These findings indicate that the aromatic ring of
phenylalanine or tyrosine is needed to discriminate the 2'- and 3'-OH
groups. Using mutant characterization and three-dimensional structure
analysis (6), the catalytic domain of T7 RNAP was separated into the T7
promoter binding region (674-752 aa), Mg2+ binding sites
(Asp-537, Asp-812), and essential regions of polymerase activity (motif
A, motif B, motif C). To analyze the mechanism of the polymerase
reaction, the interaction sites for the 2'- and 3'-OH group have been
extensively examined (3-5).
Recently, Tyr-639 in the active site has been reported to be a
discrimination site for 2'-OH groups in T7 RNAP (7). The Y639F mutant
retains DNA and RNA polymerase activities but cannot discriminate rNTP
from 2'-dNTPs. To further determine the mechanisms of the polymerase
reaction, the 3'-OH discrimination site in the elongation step needed
to be identified. However, details of the biochemical mechanism to
discriminate the 3'-OH group of rNTPs by the T7 RNAP have not been
clarified.
Until now, Met-635 on T7 RNAP has been thought to be the interaction
site of 3'-OH groups based on homology with the Klenow fragment. This
has been the case because the affinity for the rNTP molecule decreases
when mutation occurs on Met-635 (8). As mentioned above, Met-635 and
Tyr-639 were thought to be the only discrimination sites of 2'- and
3'-OH groups, respectively. They were thought to be a part of the
catalytic pocket due to their geometrical alignments.
We found that the Phe-644 and Phe-667 sites that are located downstream
of the Y639 site are important sites for interacting with the 3'-OH
group of rNTPs in the T7 RNAP. These locations are the opposite of the
location previously thought to be only one responsible for
discriminating 2'- and 3'-OH groups. These findings suggest that the T7
RNAP may interact in several ways to the 3'-OH group of rNTPs in
addition to Met-635. Our findings are in agreement with the reverse
compensation theory of 2'- and 3'-OH groups proposed by Tabor and
Richardson (5). This discrimination mechanism of the 3'-OH group seems
to have been conserved during the process of molecular evolution.
Recently, we developed a novel sequencing method (transcriptional
sequencing) using T7 RNAP. This reaction system was improved by the use
of F644Y and F667Y mutants to incorporate 3'-dNTPs very efficiently
(25).
 |
EXPERIMENTAL PROCEDURES |
Construction and Enzyme Purification of Mutant T7 RNA
Polymerases--
Mutant polymerase genes were constructed by
PCR-mediated site-directed mutagenesis (9). Mutant enzyme expression
and large scale purification have been described previously (10,
11).
Transcription Reactions--
Nucleotides and radioactive
nucleotides were purchased from Amersham Pharmacia Biotech. The
transcription assay was done in buffer containing 40 mM
Tris-Cl, pH 8.0, 8 mM MgCl2, 5 mM
dithiothreitol, 200 µM GMP, and 5 units of T7 RNA
polymerase in the presence of rNTP and template DNA at 37 °C for 15 min to 1 h. Before all of the experiments, the quantity of T7 RNAP
enzyme was calibrated, and each sample was adjusted with respect to
their protein quantities.
Relative activity and processivity were assayed by measuring the amount
of the radioactive substrates using DE81 paper binding assay as
described elsewhere (7, 12). The amounts of rNTP and template DNA are
given in the figure and table legends.
Preference of Each 3'-dNTP Incorporation--
Recognition
selectivity for each NTP/3'-dNTP was assayed as described (5). The data
was analyzed with 8% acrylamide containing 6 M urea and
using BAS 2000 analyzing systems (Fuji Photo Film Co., Ltd.).
Transcriptional Sequencing--
The sequencing strategy and
method are described elsewhere (25). Sequencing reaction mixtures
contained the PCR product of human thyrotropin-
cDNA (13) as
sequencing template, the fluorescent 3'-dNTPs (1 µM
tetramethyl rhodamine-3'-dUTP, 0.1 µM rhodamine 6G-ATP,
0.1 µM rhodamine 110-GTP, 5 µM
X-rhodamine-CTP), 500 µM GTP, 500 µM UTP,
250 µM ATP, 250 µM CTP, 10 units of
phosphatase purified from baker yeast and 25 units of T7 RNA
polymerases. The reaction was carried out at 37 °C for 1 h. The
excess of fluorescent 3'-dNTPs was eliminated by Sephadex G-50 column
(Amersham) subjected to ABI PRISMTM 377 DNA Sequencer
(Perkin-Elmer Corp.).
 |
RESULTS |
Mutation Sites of the Constructed T7 RNAP Mutants--
Fig.
1 shows the mutation sites of the
constructed T7 RNAP mutants on the primary and three-dimensional
structures (6). Bacteriophage T7 RNAP catalyzes the phosphodiester bond
formation between the 5'-phosphate group and the 3'-OH group of rNTP,
resulting in elongation of the nascent RNA chain. To understand the
sites to be mutated, we analyzed the three-dimensional structure,
paying attention to the region of helix Y(625-634 aa), loop
(635-648aa) - helix Z (649-658 aa), loop (658-684 aa) in T7 RNAP.
It has been reported that the mutants of Lys-631, Tyr-639, Gly-640,
Asp-812, and Asp-537 were characterized among the amino acid residues
existing on the protein surface (14-17). Among these residues, Asp-812
and Asp-537 were identified as being critical for binding to the metal ion (18). Lys-631, Tyr-639, and Gly-640 were located inside motif B or
in its vicinity. The Tyr-639 site is known to be important for
rNTP/dNTP discrimination (7). On the basis of the known sites
interacting with the 2'- or 3'-OH group, we hypothesized the aromatic
residues of polymerases to be 3'-OH group to be candidates for
discrimination sites. This concept is supported by reports that the 2'-
or 3'-OH discrimination sites are Phe-762 and Phe-766 of E. coli DNA polymerase I (3, 4), Phe-667 of Taq DNA
polymerase (5), Phe-155 of Moloney murine leukemia virus reverse
transcriptase (19) and Tyr-737 of mycobacterium DNA polymerase I (20).
These studies have suggested that the aromatic moiety of phenylalanine and tyrosine residues can discriminate 2'- or 3'-OH groups. We further
hypothesized that the para-H group of the aromatic ring of
phenylalanine interacts with 2'- and 3'-OH groups of rNTP, and the
para-OH group in tyrosine interacts with 2'- and 3'-H groups, with hydrogen bond formation to stabilize these complexes. This
stabilization triggers the formation of enzyme-substrate complexes to
the catalysis-mediated transition state. With this in mind, we analyzed
the aromatic residues in the 625-684 aa region in T7 RNAP and selected
Phe-644, Phe-646, and Phe-667 as important candidates for
discriminating the 3'-OH group. We then constructed the mutants of each
site: F644Y, F646Y, and F667Y. We also selected other residues on the
protein surface of the T7 RNAP catalytic domain and constructed the
mutants F733Y, F782Y, and F882Y for comparative analysis.

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Fig. 1.
Mutation sites of T7 RNA polymerase
mutant. The mutated residues and highly conserved motifs among
various DNA and RNA polymerases are indicated in this figure. The
motifs and mutated sites are located above and below the amino acid
sequence, respectively. Motif B, Motif C,
HelixY, HelixZ, and Helix AA are
located at 625-652, 805-818, 625-634, 649-658, and 684-699,
respectively. The series of T7 RNA polymerase mutants designed in this
study consisted of F644Y, F646Y, F667Y, F733Y, F782Y, F882Y, and
Y639F.
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By sequencing several clones obtained by PCR, we found that our
sequence was different form that present in the GenBank data base.
Additionally, the sequence of T7 genome matched our data. For example,
proline 665 was substituted by leucine in the region estimated for the
rNTP binding. Since at site 665 there may be leucine in the sequencing
at the T7 genome, we considered T7 RNAP with leucine at the 665 site to
be the wild-type T7 RNAP. We also constructed an add-back mutant
substituting with proline at Leu-665 (L665P) for comparison with
respect to processivity and 2'- or 3'-OH discrimination efficiencies.
No differences were noted between the L665P and wild type (data not
shown).
Mutational Effects on the Biological Activity of the Wild
Type--
We analyzed the effects of processivity, relative
activity,
and 3'-dATP incorporation efficiencies in
each mutant polymerase (Fig. 2, Table I, and Table
II). Table I shows the correlation between the disruptive effects of the relative activity by the mutations. Among the constructed mutants, F882Y showed marked reduction
in relative activity to the wild type. The Phe-882 site has been
reported to be the contact site with the base of rNTP during the
elongation process. The marked decrease in F882Y activity is thought to
occur because the mutations affected the ability for extension,
resulting in disruption of the elongation reaction (21). Some mutants
show reductions in transcriptional activity, but these reductions were
not drastic because the substitution of phenylalanine for tyrosine is a
small conformational change on T7 RNAP.

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Fig. 2.
Processivity of T7 RNA polymerase
mutants. The assay method used in this study was described
previously (12). The processivity of each RNA polymerase was measured
by the incorporation of [ -32P]UTP using a circular DNA
as the template. The reaction mixture was 40 mM Tris-Cl, pH
8.0, 8 mM MgCl2, 5 mM
dithiothreitol, 200 µM GMP, and 5 units of T7 RNA
polymerase in the presence of 250 µM NTP and 0.2 µCi of
[ -32P]UTP (3000 Ci/mmol). In this reaction mixture, 10 µmol of closed circular reeler cDNA clone was used as the
template. The final product was subjected to electrophoresis on 8%
polyacrylamide gel containing 6 M urea and
autoradiographed. 1st lane, wild type; 2nd lane,
F644Y; 3rd lane, F667Y; 4th lane, Y639F;
5th lane, F646Y; 6th lane, F733Y; 7th lane
, F782Y; 8th lane, F644Y/F667Y; 9th lane,
F882Y.
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Table I
Relative activity of T7 RNA polymerase mutant
The relative activity of each RNA polymerase mutant was measured as the
incorporated rate of [ -32P]UTP. The reaction was performed
in the presence of 250 µM each rNTP using the plasmid DNA
digested by PvuII as the template. ND indicates that no
activity was detected. Total reaction aliquots were spotted onto DE81
paper, and after washing, the retained radioactivity of DE81 paper was
counted.
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Table II
Relative efficiences of recognizing the 3'-OH group of ribonucleotides
as substrate in each mutated T7 RNAP and wild type
For the template DNA and reaction conditions, see Table I. 3'-dATP
concentrations were 100 µM. The experiment was
triplicated. The value in this table represents the average of the
relative reduction of the [ -32P]UTP incorporation rate by
each RNAP in the presence of 100 µM 3'-dATP. The
wild-type RNAP activity is normalized to 1.000.
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Fig. 2 shows the results of determination of mutant processivities,
using closed circular reeler cDNA, which is the transcript responsible for reeler mouse mutant clone (22) as the template. Processivity values were determined from differences in the length of
transcripts. We could not find any significant difference in the
processivity of F667Y, F646Y, and Y639F mutants compared with the
wild-type polymerase. No other changes were observed for the processivity of other mutants.
Specific Recognition of the 3'-OH Group by Phe-644 and
Phe-667--
As shown in Table II and Fig.
3, the Phe-644 and Phe-667 residues
specifically recognizing the 3'-OH group were determined by analyzing
the 3'-dATP incorporation rate in polymerases. Table II shows the
3'-dATP incorporation efficiencies to the elongation chain with plasmid
DNA pBS linearized with PvuII as the template. The different
properties for recognizing the 3'-dATP substrate among the mutants were
determined by analyzing the inhibitory effects by 3'-dATP on the
extension of the elongation chains. This assay shows that the mutations
of F644Y and F667Y affect the ability of the enzyme to recognize the
structural differences between rNTPs OH and the 3'-dATP H groups. Thus,
3'-dATP is a good substrate for F644Y and F667Y mutants.

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Fig. 3.
Chain termination study for the preference of
the incorporation of 3'-dNTPs and rNTPs in F644Y and wild-type T7 RNA
polymerase. Template DNA and electrophoresis conditions are given
in Fig. 2. Each lane also had wild-type (WT) and
F644Y mutant RNAP (lanes 1-5 and 6-10, respectively).
3'-dATP concentrations in lanes 1 and 6, 500 µM; lanes 2 and 7, 100 µM; lanes 3 and 8, 50 µM; lanes 4 and 9, 2.5 µM; lanes 5 and 10, 1 µM.
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In comparison, the substitution of tyrosine at 646 reduced the
incorporation rate of 3'-dATP. With the F644Y and F667Y mutants, the
incorporation rates of 3'-dATP differed and the specificity for
recognizing the 3-OH group was better for the F644Y than for the F667Y
mutant. This shows that the mechanism for recognizing the 3'-OH group
differs between the two residues.
Fig. 3 shows the quantitative comparison of inhibitory effects to the
transcription of mutant and wild-type polymerases when the 3'-dATP was
added to the transcriptional reaction using the closed circular reeler
cDNA clones as the template (22). The incorporation rates of
wild-type polymerase for 3'-dATP as the substrate were clearly lower
than those of F644Y. As shown in Table II, this mutant increases the
3'-dATP incorporation rates. Whereas transcripts produced by the
wild-type enzyme extended almost to the terminal end of the template,
most transcripts produced by F644Y polymerase terminated at
approximately 100 base pairs, and the full-length transcript yield
produced by F644Y decreased to a few percent of all transcripts
produced (Fig. 3). Similar results were found with F667Y (data not
shown). These results indicate that Phe-644 and Phe-667 are important
sites for 3'-OH group discrimination in T7 RNAP. In a similar assay
system using E. coli DNA polymerase I, Taq DNA
polymerase, and T7 DNA polymerase, effects similar to those in Fig. 3
were reported by Tabor and Richardson (5). In our examination, F644Y
and F667Y did not incorporate 2'-dNTP. This indicates that Phe-644 and
Phe-667 might not be involved in the 2'-OH recognition (data not
shown).
Recently, Tyr-639 residue has been reported to be a discrimination site
for 2'-OH groups in T7 RNAP (7). In this work, we also examined whether
or not Y639F is a discrimination site for 3'-OH group of rNTPs. As a
consequence, the Y639F mutant showed no differences in the
incorporation rate between 3'-dATP and NTP (Table II), as reported
recently by Huang et al. (23). This mutant also showed no
changes in its ability to discriminate and incorporate 3'-dCTP,
3'-dGTP, 3'-dUTP substrate (data not shown).
Recognition Selectivity of F644Y and F667Y for 3'-dNTP and
rNTP--
Table III shows the
differences of 3'-dNTP recognition rates among the wild type and the
mutants. We determined the inhibitory effects on transcription by
adding 3'-dATP at various concentrations and by using the closed
circular reeler cDNA clone as the template. We found that the
incorporation efficiencies of 3'-dNTP by F644Y and F667Y were affected
by the structural differences in their base moieties. However,
activities of 3'-dNTP incorporation for F644Y and F667Y were >100-fold
greater than the wild-type polymerase.
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Table III
Relative selectivity of F644Y, F667Y/L665P, and wild-type T7 RNA
polymerase for 3'-dNTPs and rNTPs
The rates of each 3'-dNTP incorporation to rNTP are compared for F644Y
and F667Y mutants and wild-type T7 RNA polymerase. The template and
reaction buffer are given in Fig. 2 and Table I, respectively. 3'-dNTPs
concentrations were 500 µM and 250 µM.
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The Tyr-639 mutant was similar to its wild-type T7 RNAP with respect to
3'-dNTP discrimination (data not shown). The scale of the effect of
3'-dNTP incorporation on F644Y and F667Y activity was 3'-dUTPs > 3'-dCTPs > 3'-dATP > 3'-dGTP. The F644Y mutant led to more
efficient incorporation than the F667Y mutant with respect to the
discrimination rate of 3'-dNTP versus rNTP. The relative
affinity among 3'-dNTP and rNTP in the mutants was >100-fold greater
than in comparison to the wild type. Similar findings were reported for
E. coli DNA polymerase I (5). In consideration of all these
findings, we suggest that Phe-644 and Phe-667 are the key residues in
3'-OH discrimination.
Effect of F644Y and F667Y Mutants on Transcriptional
Sequencing--
We recently developed a novel sequencing method
(transcriptional sequencing) to resolve the problems encountered in
existing sequencing methods using DNA polymerase (25). Transcriptional sequencing offers the advantages of a high throughput, time savings, and automation for genome projects and clinical diagnosis. In transcriptional sequencing, the extension reaction is carried out with
T7 RNAP, in contrast to existing methods that use T7 DNA polymerase or
Taq DNA polymerase (24). The method being reported on (25)
is based on the chain termination reaction, performed by using 3'-dNTPs
as the terminator instead of dideoxy-NTPs in DNA polymerase-based
sequencing. In the DNA chain termination reaction method, native
Taq DNA polymerase had the problem of imbalance of
incorporation efficiencies for dideoxy-NTPs on the elongation chain,
leading to a loss of uniformity in the sequencing ladder, and
consequently obtained signals that were difficult to analyze. Recently,
these problems were overcome by thermo sequenase, which produces a
better uniformity of incorporation (24). Transcriptional sequencing
using F644Y and F667Y overcame problems in a similar fashion to those
reported by Tabor and Richardson (5). This improvement of the
sequencing pattern can be seen from Fig.
4, which presents the sequencing analysis
of the human thyrotropin-
cDNA (13) PCR product by
transcriptional sequencing with F644Y in comparison with its wild type.
The use of F644Y mutant allowed us to obtain uniform and long sequence
signals. These signals were similar to those obtained with the F667Y
mutant (data not shown). In Fig. 4, the sequence signals indicated as
arrowheads are high background signals, which did not change. Only the
intensity of the correct signals increased for the F644Y mutant, thus
yielding results of higher accuracy and confidence levels when the
F644Y and F667Y mutants were used (25).

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Fig. 4.
Electropherogram of transcriptional
sequencing using F644Y mutant. The reaction was carried out with
PCR product of human thyrotropin- cDNA (14) and F644Y mutant or
wild-type (WT) T7 RNA polymerase. The figure shows the
G-signals. Arrowheads show the high background signals of
wild-type T7 RNA polymerase. The precise protocol will be reported
elsewhere (25).
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 |
DISCUSSION |
We have shown here that the Phe-644 and Phe-667 sites on T7 RNAP
are important for 3'-OH group discrimination. In the protein structure,
Phe-644 is located in the region of the loop (635-647 aa) between
helix Y(624-634 aa) and helix Z(648-658 aa) in the catalytic domain.
The loop between helix Y and helix Z contains important residues
discriminating the rNTP molecule because Tyr-639 is located in this
loop. The Phe-667 site is located in the region of the loop (659-684
aa) between helix Z (648-658 aa) and helix AA685-700 aa).
Three-dimensional analysis suggests that these loops are positioned in
the neighborhood of the rNTP molecule. Thus, Phe-644 and Phe-667 in the
two loops seem to closely interact to the 3'-OH group of the elongating
rNTP molecule during the 3'-dNTP discrimination phase of the
reaction.
Experimental results using in vitro mutagenesis have shown
that Met-635 (8) and Tyr-639 (7) interact with 3'-OH and 2'-OH groups,
respectively. The geometries of Met-635 and Tyr-639 are very important
to interact with 3'-OH and 2'-OH groups. However, our data indicated
that the geometrical alignment of T7 RNAP and the rNTP molecule also
lies downstream of Tyr-639. This geometrical alignment to Phe-644 and
Phe-667 is the opposite of that between Met-635 and Tyr-639. These
findings suggest that in T7 RNAP, there are at least two ways to access
the 3'-OH group. This data agrees with the reverse compensation theory
of the OH and H groups (5) on the polymerase-substrate complex,
because the phenylalanine on T7 RNAP was found to be a very good target
for discriminating the 3'-OH group. Thus, this mechanism, which makes
use of the aromatic moiety, is conserved in the process of molecular
evolution of polymerases.
Mutation of Phe-644 and Phe-667 to tyrosine increased the 3'-dNTP
incorporation, but F644Y showed better incorporation than F667Y and
Phe-644, which is located in the neighborhood of the Tyr-639, which was
reported to be the 2'-OH discrimination site (7). This suggests that
the Phe-644 directly interacts with the 3'-OH group, whereas the
Phe-667 has indirect discrimination activity, such as that via metal
ions. In addition, mutation of both sites to tyrosine (F644Y/F667Y)
increased the incorporation rate of 3'-dATP better than that for F644Y
or F667Y mutants. As shown in Tables II and III, these results suggest
that Phe-644 and Phe-667 residues access the 3'-OH group from different
directions.
Until now, Met-635 on T7 RNAP was considered to be an important site
for interacting with the 3'-OH group because of its sequence homology
to E. coli DNA polymerases I and the fact that mutation of
Met-635 to alanine on T7 RNAP causes a large increase in
Km values for rNTP (8). However, these results could
not be confirmed directly for 3'-OH discrimination; only the mutant of
this site, Met-635, has low affinity to rNTP, and the direct evidence
has not yet been shown that Met-635 recognizes 3'-OH group
directly.
We found that Phe-644 and Phe-667 mutated to tyrosine, F644Y and F667Y,
in order to incorporate 3'-dNTPs on the order of 100-270-fold greater
in comparison to the wild type. Similar results were obtained when the
3'-OH discrimination residue in DNAP I (Phe-762) was mutated (5).
In this report, we have shown that two sites in T7 RNAP (Phe-644,
Phe-667) are key residues for 3'-OH discrimination. This suggests that
other DNA polymerases, RNA polymerases, and reverse transcriptases may
have direct or indirect sites for 3'-OH discrimination and such
residues have not yet been identified. Our observations suggest that
further work on elongation analysis and 2' and 3'-OH discrimination
mechanisms is required.
By using the F644Y and F667Y mutants, highly accurate and
confident signals were obtained because of the lower discrimination between rNTPs and 3'-dNTPs. As reported (25), transcriptional sequencing has advantages for the genome field.
 |
ACKNOWLEDGEMENTS |
We thank Mari Itoh for technical assistance,
Naoko Kazuta for secretarial assistance, and Dr. Masayoshi Itoh for
helpful discussions.
 |
FOOTNOTES |
*
This research was supported by Special Coordination Funds
and a research grant for the Genome Exploration Research Project from
the Science and Technology Agency of the Japanese Government and a
grant-in-aid for Scientific Research on Priority Areas and Human Genome
Program from the Ministry of Education and Culture, Japan (to Y. H).
This study was supported in part by Core Research for Evolutional
Science and Technology (CREST) from Japan Science and Technology
Corporation.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 should be addressed. Tel.:
81-298-36-9145; Fax: 81-298-36-9098; E-mail:
yosihide{at}rtc.riken.go.jp.
1
The abbreviations used are: RNAP, RNA
polymerase; aa, amino acid; PCR, polymerase chain reaction.
 |
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