From the Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea
Received for publication, September 20, 2000
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ABSTRACT |
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A series of active elongation complexes of the
phage T7 RNA polymerase were obtained through stepwise walking of the
polymerase along an immobilized DNA template. Transcripts were
radiolabeled at the 16th to 18th residues, and a photocross-linkable
4-thio-UMP was separately incorporated at the 22nd, 24th, 32nd, and
38th residues. Such complexes (up to 51 nucleotides) produced by
the incorporation of one nucleotide at a time were isolated and
individually subjected to long wave UV cross-linking. Only when the
cross-linker was positioned at the 3'-end ( The structural organization of transcription elongation complexes
has been well characterized with histidine-tagged Escherichia coli RNA polymerase arrested at specific positions on templates (1). Macromolecular interactions among DNA, RNA, and E. coli RNA polymerase in the elongation complexes have been mapped (2-5). A
double clamp model has been proposed for elongating E. coli RNA polymerase where DNA entry and RNA exit sites are directed opposite
to each other in the same region (5). Experimental evidence (6, 7) also
is consistent with this mechanism for RNA polymerase action.
The T7 RNA polymerase, a single-subunit protein of 99 kDa, is capable
of initiating transcription promoter-specifically, elongating with high
processivity and terminating in the absence of additional factors. The
mechanism of initiation and termination of T7 RNA polymerase has been
studied extensively because of its simplicity of structure and
reaction system. On the other hand, elongation has been little studied,
mainly because a series of stable and active ternary elongation
complexes have not been obtained with the T7 RNA polymerase.
The overall three-dimensional structures of T7 RNA polymerase complexed
with an open promoter (8) and a transcribing initiation complex (9) are
similar to that of free enzyme (10). Some regions, including the active
site, change into a more compact structure in the initiation complex.
Structural information on elongation complexes with a growing
transcript has not yet been determined.
Several lines of evidence have suggested that the structure of the T7
RNA polymerase ternary complex changes when it proceeds from the
initiation phase to the elongation phase. Both protease sensitivity
(11) and the protected region of the DNA template strand in
footprinting with methidiumpropyl-EDTA-Fe(II) greatly change (12, 13)
during the switch from initiation to elongation. The length of the
DNA-RNA hybrid also changes from 2-4
bp1 in the initiation complex
(11) to 7 bp in an arrested elongation complex (13). However, the
macromolecular interactions and the structures of the active ternary
elongation complexes have not yet been examined.
In this study, we obtained a series of active and stable elongation
complexes of the T7 RNA polymerase prepared by walking the enzyme along
an immobilized DNA template. To probe RNA-protein interactions in
ternary elongation complexes, a 4-thio-UMP was incorporated into the
nascent transcripts and used to photocross-link RNA to the polymerase.
Our cross-linking results suggest that the RNA runs from the active
site toward a C-terminal region in the finger domain, rather than an
N-terminal region near the thumb domain, in active elongation complexes
of the T7 RNA polymerase.
DNA Template--
The 216-bp BglII fragment of pET3
(14) containing a T7 promoter and the T Stepwise Walking of T7 RNA Polymerase--
The biotinylated
templates (30 pmol) bound to streptavidin beads were incubated with 60 µl of an equimolar T7 RNA polymerase (United States Biochemicals) in
transcription buffer A of 40 mM Tris-HCl, pH 7.9, 6 mM MgCl2, 100 mM KCl, and 10 mM dithiothreitol at 37 °C for 5 min. The transcription
reaction was started by adding 60 µl of a ribonucleotide mixture to
final concentrations of 0.05 mM ATP, 0.05 mM
GTP, and 0.005 mM CTP and continued for 20 min to produce a
15-mer RNA complex, TEC15. After five washes with 0.5 ml of
transcription buffer B consisting of 40 mM Tris-HCl, pH
7.9, 6 mM MgCl2, and 100 mM KCl,
the beads were resuspended in 120 µl of buffer B containing 0.33 µM [
A photo-reactive analog of UMP, 4-thio-UMP was incorporated into the
+24 site with incubation in a 100-µl mixture of 5 µM 4-thio-UTP and transcription buffer C consisting of 40 mM
Tris-HCl, pH 7.2, 6 mM MgCl2, and 100 mM KCl at 37 °C for 2 min. The
4-thiouridine-incorporated radioactive TEC24 was elongated further with
suitable ribonucleotides along the template sequence to TEC25, TEC26,
TEC27, TEC30, TEC31, TEC32, TEC34, TEC36, TEC38, TEC40, TEC41, TEC43,
TEC44, TEC47, TEC48, and TEC51 in progressively smaller reaction
volumes. A 5-µl portion of each 15-µl aliquot was mixed with 10 µl of gel loading buffer of 10 mM EDTA, 12 M
urea, and 0.1% (w/v) bromphenol blue and was loaded onto an 8 M urea, 10% polyacrylamide gel.
Photocross-linking of Elongation Complexes--
A 10-µl
portion of each elongation complex aliquot was transferred to a 1.5-ml
Eppendorf tube placed on ice and was then subjected to UV irradiation
for 20 min using a 360-nm lamp UVGL-25 (Ultraviolet Products), as
described previously (3). Each irradiated sample was mixed with 2.5 µl of gel loading buffer consisting of 60 mM Tris-HCl, pH
6.8, 25% (w/v) glycerol, 2% (w/v) SDS, 14.4 mM
Mapping of the Trypsin Cleavage Site and RNA-linked
Sites--
The trypsin cleavage site of elongating T7 RNA polymerase
was mapped by using N-terminal histidine-tagged T7 RNA polymerase that
was produced in E. coli from pKK-HisT7 (16). The plasmid was
constructed by Hoseok Song and Changwon Kang (16) who transferred the
T7 polymerase gene from pAR1219 into the XhoI site of
pET-tac, derived from pET15b (Novagen). The 21-mer
transcript-containing elongation complex of the His-tagged polymerase
was obtained by walking and was partially digested by trypsin. The
polypeptides separated by SDS-PAGE were transferred to nitrocellulose
membrane by electroblotting. The membrane was probed with a monoclonal His-1 antibody against poly(His) (Sigma) and incubated with anti-mouse immunoglobulin G conjugated to alkaline phosphatase (Sigma) as described previously (17).
To map the cross-linked sites the RNA-linked elongation complexes were
partially digested with trypsin (18). They were also denatured and
partially digested by hydroxylamine (3), 2-nitro-4-thiocyanobenzoic acid (19), and cyanogen bromide (20), as described previously. Cleavage
products were isolated by an 8-15% gradient or a 10% polyacrylamide/SDS gel in a Tris-glycine buffer, pH 8.3 of 25 mM Tris, 192 mM glycine, and 0.1% SDS.
Prestained standards of different protein sizes were purchased from
Bio-Rad Laboratories.
Stepwise Walking of T7 RNA Polymerase and Cross-linking of
Elongation Complexes--
Productive elongation complexes of the phage
T7 RNA polymerase were prepared by walking of the polymerase along an
immobilized template DNA. The 5'-end of template strand was immobilized
by biotin-streptavidin conjugation. The template used here had the same
T7 transcription unit as pET3, except for TTT at residues 19-21 from
the initiation site (Fig. 1A)
and allowed for production of 15-mer RNA in the absence of UTP in
transcription reactions.
The 15-mer RNA in the complex, called TEC15, was extended to an 18-mer
by the incorporation of [
The transcription complexes from TEC24 to TEC47 in which the RNA
contained a photocross-linker and three radioactive residues were
irradiated with
These experiments were repeated with the complexes where the
photocross-linker was separately incorporated at the 22nd, 32nd, and
38th residues of the transcripts. When the cross-linker was at the 22nd
residue, the 22-mer and 30-mer RNA, respectively in TEC22(sU22)
and TEC30(sU22), produced the most predominant cross-linking (Fig.
2A). Likewise, with the cross-linker at the 32nd residue, TEC32(sU32) and TEC40(sU32) were cross-linked to the greatest extent (Fig. 2C). Also with the cross-linker at the 38th
residue, TEC38(sU38) produced the most pronounced cross-linking (Fig.
2D). TEC46(sU38) could not be isolated because
AMP was consecutively incorporated into the 45th to 47th
residues. TEC44(sU38) and TEC47(sU38), however, produced only a
residual level of cross-linking like the other complexes. Thus, the
polymerase-linking sites of transcripts were the same regardless of RNA
length and cross-linker position.
Mapping of Two RNA-linked Sites to the C-terminal One-Third Region
Using Trypsin and Hydroxylamine--
The two RNA-linking sites of the
elongating polymerase might be different from each other, because they
are separated by 8 nucleotides on elongating transcripts. The two
sites, named 3'-end and
Native T7 RNA polymerase is susceptible to proteolytic cleavage by
trypsin. Initial cleavage occurs near Lys172 or
Lys180 under mild conditions and results in a nicked RNA
polymerase consisting of N-terminal 20-kDa and C-terminal 79-kDa
fragments (18). It was not clear, however, if the cleavage site was
still the same in the elongating RNA polymerase. To address this issue, the T7 RNA polymerase that was histidine-tagged at the N terminus was
used for walking to TEC24. When such an elongation complex was digested
by trypsin under mild conditions, only the 20-kDa fragment was detected
on a Western blot with an anti-histidine antibody (data not shown).
Thus, trypsin cleaves the T7 RNA polymerase initially at the N-terminal
one-fifth location in an elongation complex similar to free enzyme
under the conditions used (Fig. 3).
Partial digestion of the RNA-linked complexes TEC24(sU24) and
TEC32(sU24) with trypsin yielded only one radiolabeled fragment whose
size was about 90 kDa (Fig.
4A). Molecular masses of the 24- and 32-mer RNA are ~7,600 and 10,100, respectively. Thus, RNA was
cross-linked to the C-terminal 79-kDa fragment of the polymerase, but
not to the N-terminal 20-kDa fragment in both complexes. When a
4-thiouridine was incorporated in the transcripts at the 22nd, 32nd,
and 38th residue positions, the results of trypsin cleavage mapping
were the same (data not shown).
Hydroxylamine was then used to map the RNA-linking sites of elongating
T7 RNA polymerase. There are only two hydroxylamine cleavage sites on
the T7 RNA polymerase (Fig. 3), but total digestion should produce
three polypeptides of approximately the same size (32, 34, and 33 kDa
from the N-terminus). Thus, SDS-PAGE analysis of partial cleavage
fragments of 32-34, 66-67, and 99 kDa would not identify the linked
region. However, partial cleavage of the RNA-linked 79-kDa fragment
would produce different sets of radiolabeled fragments, depending on
which of three regions is linked to the RNA.
The elongation complex TEC24(sU24), containing the cross-linker at the
24th residue of transcripts, was analyzed. The radiolabeled RNA-linked
79-kDa fragment was eluted from an SDS-PAGE gel and partially digested
with hydroxylamine. The fragments of (33+8), (67+8), and (79+8) kDa
were radioactive whereas those of (12+8) and (46+8) kDa did not show
any radioactivity (Fig. 4B). These results unambiguously
demonstrated that the 3'-end was cross-linked to the C-terminal
one-third region of Gly589-Ala883 (Fig. 3).
Elongation complex TEC32(sU24) was also analyzed in the same way. Only
fragments of (33+10), (67+10), and (79+10) kDa were radioactive. (The
32-mer RNA is 10 kDa.) Thus, the Fine Mapping of Two RNA-linked Sites Using Chemical
Digestions--
In mapping experiments with 2-nitro-5-thiocyanobenzoic
acid, single-hit digestion conditions were used, because there are 12 Cys cleavage targets (Fig. 3). As RNA is linked to the C-terminal one-third region, only the smallest radiolabeled fragment produced under such conditions can determine the linked region. Thus, fragment complexes smaller than about 50 kDa (for
Ser541-Ala883 peptide plus RNA) were closely examined.
In the case of single-hit digestions of TEC24(sU24), the smallest
radiolabeled fragment was of (38+8) kDa (Fig.
5, A and B, filled arrows), representing the size of the
Ser541-Ala883 region, and the (18+8)-kDa
fragment of Ala724-Ala883 was not observed.
The fragments of (46+8), (60+8), (69+8), (74+8), and (85+8) kDa were
also radioactive, possibly representing the Ala468-Ala883,
Pro348-Ala883,
Val272-Ala883,
Ile217-Ala883, and
Leu125-Ala883 regions, respectively.
Therefore, the cross-linked fragments contained the
Ser541-Cys723 region. This mapping was
supported by the results of more extensive digestion at a higher pH or
for a longer reaction time. A group of bands around 36 kDa (Fig. 5,
A and B, arrows) should not have been observed if
the cross-linker were located in the other regions, Ala724-Cys839 (13 kDa) or
Asp840-Ala883 (5 kDa). Combining the results
of hydroxylamine mapping, the 3'-end contact site is in the region of
Gly589-Cys723 (Fig. 3).
Single-hit cleavage patterns of TEC32(sU24) were different from that of
TEC24(sU24). The smallest observed fragment was of (18+10) kDa (Fig. 5,
A and B, filled arrows), reflecting the size of
the Ala724-Ala883 peptide, and a (5+10)-kDa
fragment of Asp840-Ala883 was not observed.
These results were consistent with those of more extensive digestion,
as the smallest observed fragment was of (13+10) kDa (Fig. 5,
A and B, arrow) possibly containing the 13-kDa
Ala724-Cys839 peptide. Thus, the
There are 26 potential Met sites of cyanogen bromide cleavage (Fig. 3).
When the denatured TEC24(sU24) was treated with cyanogen bromide under
single-hit digestion conditions (Fig. 5C), the smallest radioactive fragment was of (28+8) kDa for the
Thr636-Ala883 region. Thus, the 3'-end contact
site is in the Thr636-Met666 region. Likewise
the smallest fragment of TEC32(sU24) was of (21+10) kDa for
Asn697-Ala883 (Fig. 5C), suggesting
that the
Cross-links in elongation complexes TEC32(sU32) and TEC40(sU32) that
contained the photocross-linker at the 32nd residue were also mapped in
the same way. The mapping results were the same as above; the
Thr636-Met666 and
Ala724-Met750 regions, when located at Transcription elongation complexes of the T7 RNA polymerase have
been obtained either by placing psoralen cross-link site specifically
downstream from a promoter (13) or by withholding a ribonucleotide from
a transcription reaction mixture (21). These arrested or stalled
ternary elongation complexes were not subjected to further elongation.
In this study, several series of active ternary complexes of the T7 RNA
polymerase were obtained by the polymerase walking method. It was
achieved here by immobilizing biotinylated DNA templates with
streptavidin beads rather than by immobilizing the RNA polymerase.
Elongation complexes of both intact and N-terminal histidine-tagged
polymerases halted because of missing nucleotides were capable of
extending transcripts with replenishment of nucleotides.
A 4-thio-UMP was incorporated separately at four different sites (22nd,
24th, 32nd, and 38th residues) in transcripts, and four series of
elongation complexes were obtained by walking. Major photocross-links
between RNA and polymerase are observed when the cross-linker is
positioned at the 3'-end ( The upstream ( The two residues at the 3'-end and 1) of the elongating RNA
and 8 nucleotides upstream (
9), was the RNA substantially
cross-linked to the polymerase, regardless of how far it was from the
5'-end of the transcripts. Linkage of the 3'-end residue was mapped to the Thr636-Met666 region, which contains
nucleotide-binding sites. The
9 residue was cross-linked to the
Ala724-Met750 region rather than to the
N-terminal region. These two contacts were maintained throughout the
elongation complexes and reveal a route of nascent RNA through the T7
RNA polymerase in elongation complexes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
terminator was inserted into
the BamHI site of pUC19 (15). GGG at position 19-21
(relative to the transcription start site at +1) was mutated to TTT to
avoid formation of an RNA hairpin structure. Transcription templates
were obtained by polymerase chain reactions using biotinylated reverse
and forward M13 primers and were immobilized by incubating with
streptavidin-coated magnetic beads (Dynal, Inc.) at room temperature
for 15 min.
-32P]UTP (3,000 Ci/mmol, Amersham
Pharmacia Biotech) and incubated at room temperature for 2 min to
obtain 32P-labeled TEC18. After four washes, the beads were
resuspended in 0.8 ml of buffer B, and a 15-µl aliquot was withdrawn.
The washed beads were then resuspended in 115 µl of buffer B
containing 0.5 µM ATP for 2 min to obtain TEC21. TEC22
and TEC23 were obtained similarly in 110- and 105-µl reaction
volumes, respectively.
-mercaptoethanol, and 0.1% (w/v) bromphenol blue and then heated to
95 °C for 5 min. It was then loaded onto an 8% polyacrylamide/SDS
gel for separation of the cross-linked and uncross-linked polymerase. The electrophoresis buffer, pH 8.3 consisted of 25 mM Tris,
192 mM glycine, and 0.1% SDS.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Walking of the T7 RNA polymerase.
A, sequence of the RNA from the 5'-end. B,
transcripts of elongation complexes with a 4-thiouridine incorporated
at the 24th residue. The sizes of RNA in the number of nucleotides
(nt) are shown at the top. The positions of the
cross-linker (sU) relative to the 3'-end ( 1) of RNA are
shown at the bottom.
-32P]UMP at three residues,
16-18. A photo-reactive analog 4-thio-UMP was then incorporated at the
24th residue. The elongation complex was still active in further
elongation after storage at 4 °C for a week. Thus, all possible
elongation complexes from TEC15 to TEC51 were produced by RNA extension
with one kind of nucleotide at a time, as partially shown in Fig.
1B.
360 nm UV. Upon separation of the cross-linked complexes from the uncross-linked RNA by SDS-PAGE, the
radiolabeled RNA was shown to be cross-linked to the polymerase throughout all the complexes at least to a low level, as shown in Fig.
2. The radioactivity of a cross-linked
complex was normalized against the total radioactivity of RNA in each
lane. The resulting relative yield of cross-linking appeared to be the
highest in two complexes, TEC24 and TEC32 (Fig. 2B). Thus,
cross-linking was most efficient when the photocross-linker (placed at
the 24th residue) was positioned at the 3'-end of transcript RNA (in
TEC24) and 8 nucleotides upstream (in TEC32).
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Fig. 2.
Cross-linked elongation complexes. A
photocross-linker was separately incorporated at the 22nd
(A), 24th (B), 32nd (C), and 38th
(D) residues. The sizes of RNA in the number of nucleotides
(nt) are shown at the top. The positions of the
cross-linker (sU) relative to the 3'-end ( 1) of RNA are
shown at the bottom. Intensely cross-linked complexes are
marked in bold at positions of the cross-linker. Complexes
without sU-incorporated transcript (O) and non-irradiated
(X) were used as negative controls.
9 contact sites here, were mapped
individually by enzymatic and chemical proteolysis of the cross-linked
elongation complexes.
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Fig. 3.
Cleavage map of the T7 RNA polymerase.
Horizontal lines indicate 883 amino acids of the polymerase.
Potential cleavage sites of trypsin, hydroxylamine (HA),
2-nitro-5-thiocyanobenzoic acid (NTCBA), and cyanogen
bromide (CNBr) are indicated by vertical bars.
Numbers on the bars indicate C-terminal amino acids of
cleavage fragments, except for those on the bottom line. The + filled and solid boxes represent the mapped regions
linked to the 3'-end ( 1) and
9 nucleotide residues,
respectively.
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Fig. 4.
Coarse mapping of the cross-linked regions in
TEC24(sU24) and TEC32(sU24). A, mapping by partial
trypsin digestion. Intact TEC24 and TEC32 were
partially treated with trypsin for indicated time periods in min and
separated in 8-15% polyacrylamide gradient SDS gels. Undigested
controls (0) are included, and bands at the
bottom are uncross-linked RNA. B, mapping by
partial hydroxylamine digestion. The RNA-linked trypsin fragments
(T) of TEC24 and TEC32 were isolated from the gel A
(C3 bands), and treated with hydroxylamine
(H) as denatured. Whole complexes (W) were also
isolated from the gel in A (C4 bands)
and treated with hydroxylamine for size standards of complexes.
Undigested controls (C) of the gel-eluted samples and
protein size standards (M) are shown.
9 residue was also cross-linked to
the same C-terminal one-third region (Fig. 3).
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Fig. 5.
Fine mapping of the cross-linked regions in
TEC24(sU24) and TEC32(sU24). Theoretical cleavage pattern with
C-terminal labeling is shown to the left with the numbers
indicating potential cleavage sites. Filled arrows indicate
the smallest labeled fragments produced under the single-hit cleavage
conditions, and regular arrows indicate the smallest from
extensive digestion. Undigested controls (C) and protein
size standards (M) are shown. A, time course in
min of proteolysis with 2-nitro-4-thiocyanobenzoic acid at pH 8.0. Numbers to the left indicate the Cys positions. B, digestion
with 2-nitro-4-thiocyanobenzoic acid at increasing pH. Digestion for 15 min was progressively more extensive at pH 8.4 than pH 8.0. C, time course in min of proteolysis with cyanogen bromide.
Numbers to the left indicate the Met positions.
9 contact
site is located between Ala724 and Cys839 (Fig.
3).
9 contact site is in the
Ala724-Met750 region. Fragments larger than
that were also observed as expected, except for those of (24+10) kDa
(for Phe667-Ala883) and (23+10) kDa (for
Ala678-Ala883). Sites at 666 and 677 were not accessible.
1 and
9 positions, respectively (data not shown).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1) of growing transcripts and 8 nucleotides
upstream (
9), regardless of how long RNA is and how far the
cross-linker is from the 5'-end (Fig. 2). Thus, two separate residues
of elongating transcripts closely interact with RNA polymerase.
4-Thiouridine appears to be indiscriminate when cross-linking to amino
acids in a protein because of its high photoreactivity (22, 23). Thus,
photoreactive groups of the other residues may not be in close contact
with the polymerase, although the nearby amino acids, if there are any,
may not have been reactive.
9) nucleotide that is near to the protein is also near
the DNA-RNA hybrid of ~7 bp, as determined from the work of Sastry
and Hearst (13). When the movement of T7 RNA polymerase was blocked by
psoralen cross-link at +36, the bottom strand of DNA between +30 and
+36 was resistant to single-strand-specific T7 endonuclease. Elongating
transcripts were previously found to interact with E. coli
RNA polymerase also at two sites (5). RNA distance between the two
sites (8-9 residues) in E. coli is similar to that in T7,
possibly reflecting the similar length of DNA-RNA hybrid within
elongation complexes. The upstream site, however, is much broader in
E. coli (about 9 nucleotide residues) than in T7 complex
(about 1 residue). Although the hybrid length is apparently similar in
T7 and E. coli RNA polymerase, stability of elongation
complexes to high salt in each case differs significantly, suggesting
that the hybrid itself does not play the major role in complex
stability. The shorter RNA binding site in T7 polymerase and/or
unlocked DNA-binding site could explain the difference in stability.
9 were found to cross-link to two
different regions of elongating T7 RNA polymerase, when mapped using
trypsin, hydroxylamine, 2-nitro-5-thiocyanobenzoic acid, and cyanogen
bromide. The RNA 3'-end linking region is between Thr636
and Met666 (Fig. 6). This is
a part of the O-helix, which has been considered to include active site
residues. A mutation of Tyr639 to Phe previously resulted
in incorporation of dNTP as well as rNTP, suggesting that it is
involved in nucleotide binding (24). In the crystal structure of the
initiation complex, the O-helix residues Met635,
Thr636, and Phe644 interact with incoming rNTP
(9). Therefore, the active site region appears to be conserved from
initiation to elongation processes.
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Fig. 6.
The route of growing RNA transcripts in the
T7 RNA polymerase. The RNA-linked regions are marked in
space-fill mode on the co-crystal structure of polymerase
and DNA (8). RNA runs from an active site within the 3'-end contact
region (green) toward the 9 contact region
(blue) rather than toward an N-terminal region (144-168,
black line) previously suggested by other investigators
(26). A part of the
9 contact region excluding the
promoter-specificity region (742-773, cyan line) is shown in
blue. Template (orange) and nontemplate
(yellow) strands of DNA are shown in ribbon mode.
The N and C termini of the polymerase are also shown.
It is surprising that the upstream RNA (9) binding region is close to
the C terminus, between Ala724 and Met750 (Fig.
6), because the N-terminal 20-kDa fragment has been thought to be
associated with RNA binding and processivity of the elongation complex.
Exogenous RNA binding was previously abolished in the C-terminal 80-kDa
fragment (18). E148A mutant was defective in RNA oligomer binding (25).
Also, based on the photocross-linking results with the photoreactive
group linked to the 5'-end of nascent RNA, Sastry and Ross (26)
proposed that regions between 144 and 168 and between 1 and 93 interact
with emerging RNA and form a solvent-accessible RNA binding channel.
Based on these previous results, Cheetham and Steitz (9) drew an RNA
path toward the thumb domain in the N-terminal region in their model
based on the crystal structure of initiation complex.
According to our results, however, elongating RNA travels from the active site region toward a C terminus proximal region rather than an N terminus proximal region (Fig. 6). Elongating and exogenous RNAs appear to bind different regions of the polymerase. In the cross-linking experiments by Sastry and Ross (26), the ternary complex was arrested with only 5-8-mer transcripts, probably had not escaped from abortive initiation cycling, and the linker used was rather long.
In our model, RNA exits in the tunnel through the finger region in the
elongation complex (Fig. 6). Thus, the RNA-exiting region is different
from the DNA-entering region. The RNA-exiting region ()
partially overlaps with the promoter-specificity loop (). This
may reflect structural continuation of template DNA strand in
initiation and elongation. More interestingly, these two regions form a
bent channel in the structure of the initiation complex (9). Recently,
DNA in the initiation complex was found to be bent 40-60 degrees
around the transcription start site (27), whereas the intrinsic bend of
the promoter was much smaller (28). The bending formed to facilitate
DNA melting for initiation would be maintained during the elongation stage.
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FOOTNOTES |
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* This work was supported by Grant 1999-1-209-002-5 from the Basic Research Program of the Korea Science and Engineering Foundation and by the Brain Korea 21 Project.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.: 82-42-869-2628;
Fax: 82-42-869-2682; E-mail: ckang@mail.kaist.ac.kr.
Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M008616200
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ABBREVIATIONS |
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The abbreviations used are: bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; TEC, transcription elongation complex.
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REFERENCES |
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