Scanning Mutagenesis Reveals Roles for Helix N of the
Bacteriophage T7 RNA Polymerase Thumb Subdomain in Transcription
Complex Stability, Pausing, and Termination*
Luis G.
Brieba,
Vijaya
Gopal, and
Rui
Sousa
From the Department of Biochemistry, University of Texas Health
Sciences Center, San Antonio, Texas 78284-7760
Received for publication, October 29, 2000, and in revised form, November 30, 2000
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ABSTRACT |
Deletions within the thumb subdomain (residues
335-408) of T7 RNA polymerase decrease elongation complex stability
and processivity, but the structure of a T7RNAP initial transcription
complex containing a 3-nucleotide RNA reveals no interactions between
the thumb and the RNA or DNA. Modeling of a longer RNA in this
structure, using a T7DNAP-primer-template structure as a guide,
suggests that the phosphate ribose backbone of the RNA contacts a
stretch of mostly positively charged side chains between residues 385 and 395 of helix N of the thumb. Scanning mutagenesis of this region
reveals that alanine substitutions of Arg391,
Ser393, and Arg394 destabilize elongation
complexes and that substitutions at 393 and 394 increase termination of
transcripts 5 or more bases in length. The
-carbons of all 3 of
these residues lie on the side of helix N, which faces into the
template-binding cleft of the RNA polymerase, and modeling suggests
that they can contact the RNA 4-5 bases away from the 3'-end. Alanine
substitutions of other residues within 385-395 do not have marked
effects on transcription complex stability, but alanine substitutions
of Asp388 and Tyr385 reduce pausing and
termination at the T7 concatemer junction. Both of these side chains
lie on the outer side of helix N, pointing away from the template
binding cleft. The thumb subdomain of T7RNAP therefore has roles both
in transcription complex stabilization and in pausing and termination
at the T7 concatemer junction.
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INTRODUCTION |
Bacteriophage T7RNA polymerase
(RNAP)1 is the best
characterized member of a widespread family of RNAPs that includes many phage RNAPs as well as the eukaryotic mitochondrial RNAPs (1, 2). With
the recent description of crystal structures of a T7RNAP-promoter complex (3) and an initial transcription complex (ITC) (4), T7RNAP presents an exceptional system for investigating the
structural mechanisms of transcription, particularly because the
transcription reaction mediated by the single-subunit T7RNAP is very
similar to the transcription reactions mediated by the more complex,
multi-subunit RNAPs (5). In particular, the mechanisms of pausing and
termination in the T7-like RNAPs and the prokaryotic and eukaryotic
RNAPs appear to be similar, because both classes of RNAPs recognize
some pause and termination sequences in common (6-8).
An especially intriguing question in the biochemistry of transcription
concerns the mechanisms of elongation complex (EC) stability. Such
mechanisms must make the EC sufficiently stable to keep the RNAP from
releasing the template or RNA while it traverses multiple kilobases of
DNA and negotiates obstacles such as DNA-bound proteins.
Simultaneously, such stability mechanisms must be responsive to
regulatory factors (9, 10) and signals encoded in the template that
cause the EC to pause or terminate (11). One of the structural features
used to meet the seemingly conflicting requirements of mobility,
stability, and responsiveness is an element that, upon DNA binding,
undergoes a conformational change that causes the polymerase to clamp
onto the template (12-18). Such elements (often dubbed "thumbs"
because of their position and the resemblance of polymerases to a
cupped right hand) appear to be a ubiquitous feature of polymerase
structure and have been shown to be important in stabilizing DNAP-DNA
complexes (19, 20). The thumb subdomain of T7RNAP is archetypal. It is
an extended structure composed of two
-helices connected by an
irregularly structured loop (13). In the apoenzyme, a portion of the
thumb that projects furthest from the rest of the polymerase is
disordered (14, 15). Upon binding template this region becomes ordered and part of it assumes helical structure (3). A role for the thumb
subdomain in stabilizing the EC was revealed by the observation that
deletions in the T7RNAP thumb subdomain decrease EC processivity (16)
and stability (17, 18).
However, although deletions in the thumb affect EC stability, no direct
interactions between the thumb and either the DNA or RNA are seen in a
T7 RNAP ITC structure (4). Because the RNA in this structure is only 3 bases in length, we modeled an extension of the RNA using the structure
of the homologous T7DNAP complexed with primer-template (21) as a
guide. This modeling suggested that the phosphate-ribose backbone of
the RNA 4-5 nucleotides away from the RNA 3'-end could contact a
segment of largely positively charged side chains between residues 385 and 395 of helix N of the thumb subdomain. We carried out scanning
mutagenesis of this stretch of residues and characterized the effects
of the mutations on RNAP activity. Our results reveal two distinct
functions for this helix. The side chains of Arg391,
Ser393, and Arg394 contribute to TC stability,
apparently through interactions with the RNA 4-5 bases away from the
3'-end. The side chains of Tyr385 and Asp388
are not important for TC stability, but mutation of these residues reduces pausing and termination at the T7 concatemer junction, a
critical step in T7 phage maturation.
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EXPERIMENTAL PROCEDURES |
Mutant RNAPs were constructed using the
CLONTECH Transformer site-directed mutagenesis kit
and the pDPT7 plasmid (22) and followed the manufacturer's
instructions. Mutant and w.t. enzymes were expressed and purified as
described as described (22). Proteolytically nicked RNAP was prepared
by incubating purified T7RNAP with whole Escherichia coli
cells as described by Muller et al. (23). Transcription
reactions were run by mixing RNAPs and templates at twice the desired
final concentrations (as specified in figure legends) in 40 mM Tris, pH 8.0, 10 mM NaCl, 10 mM
MgCl2, and 5 mM dithiothreitol. After a 5-min
preincubation, reactions were initiated by adding an equal volume of
NTPs at twice the desired final concentration (typically 0.5 mM unless otherwise indicated in figure legends) in 40 mM Tris-HCl, pH 8.0, 10 mM NaCl, 10 mM MgCl2, and 5 mM dithiothreitol.
Transcripts were labeled by including 10 µCi/µl of
[
-32P]GTP (800 Ci/mM). Reactions were
incubated at 37 °C, unless otherwise indicated, and reaction
aliquots were taken at the times indicated in individual figures and
stopped by addition of an equal volume of 95% formamide, 25 mM EDTA, 0.01% xylene cyanol, and the RNA components were
resolved by electrophoresis in 20% polyacrylamide, 1% bis-acrylamide,
6 M urea gels in 1× TBE. Analysis of the resolved transcripts was carried out with a Molecular Dynamics Phosphorimager. RNase T1 treatment of halted ECs was carried out by first forming halted ECs on linearized pPK10 (18) in reactions containing 0.5 mM ATP, 0.5 mM GTP, 0.05 µM
[
-32P]UTP and 0.1 mM 3'-dCTP (the +1 to
+16 transcript sequence from pPK10 is GGGAGAGGGAGGGAUC). After a 5-min
incubation at room temperature to allow formation of the halted,
chain-terminated EC, UTP was added to 1 mM to limit
labeling of transcripts made following the first round of synthesis.
RNase T1 was then added to 0.25 unit/µl, and reaction aliquots
were taken at time points indicated in figure legends, terminated by
phenol extraction, and mixed with equal volumes of 95% formamide, 25 mM EDTA, 0.01% xylene cyanol, and resolved by
electrophoresis on 20% polyacrylamide, 1% bis-acrylamide, 6 M urea gels in 1× TBE.
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RESULTS |
Mutant Construction--
Fig.
1A shows the structure of a
T7RNAP ITC (4). Superimposed on this structure are the first 4 nucleotides of the primer from a T7DNAP primer-template complex (21).
Superposition was carried out by maximizing the alignment of the main
chain atoms of the active site asparates of each enzyme (T7 RNAP
Asp537/Asp812 and T7DNAP
Asp475/Asp654). When this is done the first 3 nucleotides of the primer from the T7 DNAP structure align with the 3 RNA nucleotides in the T7RNAP ITC, and the fourth primer nucleotide
approaches a cluster of mostly positively charged side chains (residues
385-395) on helix N of the thumb subdomain. There are two models for
the structure of the RNA-DNA hybrid in the T7RNAP TC. A model similar
to one previously developed for E. coli RNAP and polymerase
II (24) proposes that the RNA in the T7RNAP EC separates from the
template after forming a 3-base pair hybrid and passes between the
thumb and the N-terminal domain as suggested by the green
arrow in Fig. 1A (4). Another model proposes that the
RNA forms an 8-10-base pair hybrid with the template (25). Although
the paths of the RNA in these competing models ultimately diverge, both
paths lead the RNA 4-5 nucleotides away from the 3'-end past the
385-395 segment of helix N. We therefore constructed the mutations
shown in Fig. 1B and characterized how these mutations
affect RNAP function, TC stability, and termination. A deletion
mutation (ThDel) that removes residues 359-381 in a part of the thumb
subdomain that is disordered in the apoenzyme (14, 15) but that becomes
ordered upon binding DNA (3) was similarly characterized, as was an enzyme that was proteolytically cleaved between residues 172 and 173 but in which the separate fragments remain associated ("nicked" RNAP) (23, 26).

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Fig. 1.
A, structure of a T7RNAP ITC (4)
(Protein Data Bank code 1QLN). The template strand is gray,
the nontemplate strand is purple, and the RNA is
green. Four nucleotides of DNA from a T7DNAP-primer-template
complex (Ref. 21; Protein Data Bank code 1T7P) are shown in
red. The primer/RNA nucleotides are numbered with respect to
the 3'-nucleotide (numbered 1). A previously proposed exit
path for the RNA (4) is indicated by the green arrow. The
amino acids targeted for mutagenesis are in magenta, and the
segment deleted in the ThDel mutant is blue. B,
alignment of part of the T7RNAP thumb subdomain sequence with the T3,
SP6, and K11 phage RNAP sequences. Positions at which amino acids are
conserved in at least three of the sequences are indicated with an
asterisk. The element that is disordered in the T7RNAP
apoenzyme is indicated along with the location of the deletion in the
ThDel mutant. The amino acids targeted for mutagenesis are
highlighted in red, and the substitutions made are indicated
in cyan, above the T7RNAP sequence.
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The Mutations Leave Catalytic Activity Intact but Reduce
Processivity Starting at an RNA Length of ~5 Bases--
Fig.
2A shows the transcripts
obtained in continuous initiation assays with the mutant enzymes using
a template that generates a 59-base run-off transcript from a class III
T7 promoter. The figure shows a reaction that was run for 20 min.
Reactions run for 5 or 10 min exhibit fewer total transcripts but
similar proportions of short and run-off transcripts. The short (2-9
bases) and run-off transcripts are made in large molar excess of the
amount of template or RNAP, indicating that the transcripts are being
continuously synthesized and released into solution. All of the enzymes
are active and synthesize run-off transcripts at levels similar to the
w.t. enzyme. However, some of the mutant enzymes show an increase in
the proportion of short transcripts, indicating an increase in
termination during initial transcription. Inspection of Fig. 2A reveals that the increases in termination vary as a
function of transcript length. S393A, R394A (lanes 11 and
12), and the glycine substituted enzymes (lanes
14-17) show increased termination for RNAs >4 bases in length,
whereas the ThDel mutant (lane 2) shows the greatest
increases in termination for RNAs >7 bases in length. The increases in
termination at 5, 6, 8 or 9 bases also increase the amounts of shorter
(2-4 bases) transcripts made because, following release of a
transcript, the RNAPs reinitiate. As a consequence, an increase in
termination at any point during initial transcription will increase the
total number of initiation events and abortive transcripts. However, if
the percentage of termination for each RNA is quantified as described
in the legend to Fig. 2B, we find that the increases in
termination with the mutant enzymes are limited to RNAs >4 bases in
length.

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Fig. 2.
A, transcript patterns for the indicated
RNAPs [30 nM] using HindIII-cut pT75 (42) [10
nM] as template. Lengths of the run-off (59-mer) and short
transcripts (2-9-mers) are indicated. The 1 to 17 promoter
sequence is that of a class III T7 promoter, and the initially
transcribed sequence from +1 to +10 is GGGAGACCGG. B,
relative termination (termination by the w.t. enzyme is assigned a
value of 1) for the indicated RNAPs as function of RNA length;
percentage of termination for RNA of length n = (molar
amount of RNA of length n)/(amount of RNA of length n).
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The Mutations Increase Dissociation of 6-mers, but Not 2-mers, from
the ITC--
The observed increases in termination during initial
transcription could be due to a decrease in the rates of transcript
extension and/or to an increase in the rates of transcript
dissociation. To determine whether the mutations affect transcript
dissociation, we measured the steady-state rates of transcript
synthesis in reactions in which synthesis was limited to 2-mer (GG) or
6-mer (GGGAGA) production. Presteady-state and steady-state analyses carried out under conditions similar to those used here have shown that
steady-state rates of abortive transcript synthesis are limited by the
rates of transcript dissociation (27-30), so that destabilization of
the complex leads to an increase in the rate of abortive
transcript synthesis (30, 31). In the 2-mer reaction the mutants all displayed synthesis rates similar to the w.t. enzyme (Fig.
3, A and C),
indicating that the mutations are not increasing 2-mer dissociation.
However, when we measured 6-mer synthesis we found that the mutants
that displayed increased termination during initial transcription also
showed increased rates of 6-mer production (Fig. 3, B and
D), indicating that these mutations increased the rate of
6-mer dissociation from the complex.

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Fig. 3.
A and B, representative data
for reactions with the w.t. and a mutant RNAP in which transcript
extension was limited to 2 (A) or 6 (B) bases by
inclusion of only GTP or GTP and ATP, respectively, in the reaction.
Reaction time points (min) are indicated. [template] = 10 nM, [RNAP] = 30 nM. The template in
A was a synthetic promoter comprised of a template strand
extending from 17 to +5 annealed to a nontemplate strand extending
from 17 to 5. The 17 to 1 sequence corresponded to a class III
promoter, and the sequence from +1 to +5 was GGACU. The template in
B was supercoiled pT75. C, relative rates of
2-mer synthesis for all the enzymes in reactions like those shown in
A. The rate of the w.t. enzyme was assigned a value of 1.0 and corresponds to 1.2 dimers/s(RNAP-promoter complex). D,
relative rates of 6-mer synthesis for reactions like those shown in
B. The w.t. rate (assigned a value of 1.0) is 0.1 6-mer/s(RNAP-promoter complex). Error bars in C
and D show the ranges from three experiments.
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The Mutant Enzymes Exhibit Fast Rates of Promoter Escape and Slow
Rates of Halted EC Dissociation--
The previous observations
indicate that some of the thumb subdomain mutations destabilize ITCs
that contain RNAs >4 bases in length. To determine whether these
mutations would also destabilize ECs, we measured the rates of
transcript synthesis in reactions in which transcript extension was
halted during elongation by omitting an NTP. We used a template (pPK5)
whose initially transcribed sequence is GGGAGGGAGGGAGACU (18), so that
a 14-mer is made when only GTP and ATP are present. We first
determined, for all of the mutant enzymes, whether the rates of 14-mer
synthesis in these reactions would be limited by the rate of halted EC
turnover or by the rate of promoter escape. Fig.
4 shows a representative experiment
measuring the rate of promoter escape for the w.t. and 5 mutants. On
the promoter used here the RNAP pauses when the RNA reaches 6 nucleotides in length (43). As measured from the decrease in the amount
of 6-mer and the appearance of the 14-mer, the w.t. enzyme (Fig. 4,
A, lanes 1-5, and B) clears this promoter and forms the halted complex at a rate of ~0.6
min
1 at room temperature. With the exception of S393G
(Fig. 4, A, lanes 21-25, and B), the
mutant enzyme rates of promoter escape are similar to that of the w.t.
enzyme. We found that all of the mutants, with the exception of S393G
and S393A (not shown), displayed rates of promoter escape similar to
that of the w.t. enzyme. The escape rates for S393G and S393A were
2-3-fold slower. Therefore, in these reactions the synthesis of one
14-mer/template occurs rapidly. However, following this initial burst,
the synthesis of additional 14-mer is much slower (Fig. 4). We
therefore conclude that following the initial burst the rate of
synthesis of the 14-mer (for all of the mutants) is limited by the rate
at which the halted EC dissociates.

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Fig. 4.
A, representative experiment measuring
the rate of promoter clearance and synthesis of a 14-base transcript on
ClaI linearized pPK5 (18) for the w.t. and five mutant RNAPs
at room temperature. Template and RNAP concentrations were
10 7 and 3 × 10 7 M,
respectively. The sequence of the promoter from 1 to 17 is that of
a class III promoter, and the initially transcribed sequence from +1 to
+16 is GGGAGGGAGGGAGACU, so that transcript extension is limited to 14 bases by inclusion of GTP and ATP only. Reaction time points are
indicated. B, data from A plotted and fit to:
A(1 e kt) where A
is equal to template concentration. Diamonds, S393G;
circles, w.t.; inverted triangles, R391G;
triangles, ThDel; squares, K392G; crossed
squares, R394G. The rates of promoter clearance (synthesis of one
14-mer per template) at room temperature were between 0.4 and 0.6 min 1 for all of the enzymes, with the exception of S393G
and S393A, whose rates were ~0.2 min 1.
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Thumb Subdomain Mutations Increase the Dissociation Rates of ECs
Halted on Supercoiled Templates--
We measured halted EC turnover in
reactions in which halted complexes were formed on pPK5. The +1 to +16
sequence of the transcript from this template is GGGAGGGAGGGAGACU. By
omitting CTP and including 3'-dCTP in reactions with pPK5, we could
form halted ECs containing a 15-nucleotide RNA that could not be
extended because it lacked a 3'-OH. This made it possible to measure
the stability of the EC in either the presence or absence of UTP so
that we could assess the effects of NTP binding on complex stability.
It has been found that EC stability is affected by template
supercoiling (17, 18), so we measured EC stability on both linear and
supercoiled templates. Representative data for the four different
reaction conditions (supercoiled or linear template; ±1 mM
UTP) are shown in Fig. 5A for
the w.t. RNAP and for four modified RNAPs that exhibit distinctive
behavior. The w.t. enzyme and the D388A mutant are seen to form stable
complexes that turn over at a rate of <0.01 min
1 under
all four conditions. The nicked enzyme forms a less stable complex;
without UTP present the nicked EC turns over at a rate of 0.1-0.2
min
1 on both the linear and supercoiled templates.
Addition of 1 mM UTP stabilizes the nicked complex and
reduces turnover to <0.01 min
1. Because GTP and ATP are
present at 0.5 mM in these experiments, it is clear that
this stabilizing effect requires an NTP that is complementary to the
template base immediately downstream of the RNA 3'-end (with a template
whose initial transcript sequence is GGGAGAGGGAGGGAUC stabilization of
complexes halted by 3'-dUMP incorporation required CTP addition; data
not shown). The complex formed by R391G is stable on the linear
template, but on the supercoiled template it turns over at a rate
similar to that of the nicked complex. Addition of UTP stabilizes the
R391G complex. Data for all the enzymes are summarized in panels
B-D of Fig. 5.

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Fig. 5.
A, representative experiment for w.t.
and three modified RNAPs measuring the steady-state rates of synthesis
of 15-mer transcripts on HindIII linearized or supercoiled
pPK5 with 0.5 mM GTP, ATP, 3'-dCTP, and with or without 1 mM UTP present, as indicated. B-E, steady-state
rates of 15-mer synthesis on supercoiled (B and
D) or linear (C and E) templates in
either the presence (D and E) or absence
(B and C) of 1 mM UTP. RNAP and
template concentrations as in Fig. 4, error bars give ranges
from three experiments. The turnover rate of 0.01/min represents an
estimated upper limit. At such a rate detection of synthesis of one
additional transcript/template following the initial synthesis of one
transcript required incubations of almost 2 h. We were concerned
both that the RNAP might be losing activity during such prolonged
incubations and that even very small amounts of contaminating RNases
might begin degrading the RNA. We therefore concluded that we could not
reliably measure turnover rates of less than 0.01/min.
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Effects of the Mutations on Termination at Class I and Class II
Terminators--
Because some of the thumb mutants affect EC stability
they might be expected to affect termination. T7RNAP terminates
specifically at two types of terminators (32-34). The class I
terminator is similar to classic E. coli RNAP intrinsic
terminators and is comprised of a sequence that can form a stable
hairpin in the RNA immediately upstream of a run of uracils (32). Class
II terminators are comprised of the sequence AUCUGUU followed by a run
of uracils (33, 34). In T7 DNA this sequence is found at the concatemer junction (CJ), and termination or pausing by T7RNAP at the CJ is
essential for phage development (35). Fig.
6A shows that none of the
mutations had marked effects on class I termination; the percentage
termination for all of the enzymes reproducibly fell in the 63-70%
range. Fig. 6B shows termination at a class II terminator.
As reported previously, the nicked enzyme fails to recognize the class
II terminator (<5% termination) (34, 35). The ThDel and Y385A mutants
also show almost complete bypass of the class II terminator (3-4% and
7-8% termination for ThDel and Y385A, respectively), whereas the
D388A mutation exhibits a substantial reduction in termination (19%
termination). The percentage of class II termination for the w.t.
enzyme and the other mutants reproducibly fell between 54 and 73%.

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Fig. 6.
Run-off transcription of
HindIII-cut pET-3A (Novagen Corp.; upper
panel) or BamHI-cut pLM44 (34) (lower
panel) templates that contain, respectively, either the T7
class I (T7-T ) (32) or class II (PTH) (33)
terminator sequences. RNAP and template concentrations were as in
Fig. 2. R.O., run-off; Term., terminator.
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Class II terminators act as both pause and termination signals.
Sequences that contain the class II CAUCUGUU recognition element (the
CJ sequence) but that lack a run of uracils act as pause sites (33). To
determine whether the thumb mutations affect pausing at such a site, we
measured the rate of appearance of run-off and paused/terminated
transcripts in continuous initiation assays with the w.t., Y385A,
nicked, and ThDel enzymes (Fig. 7). In
the case of the w.t. enzyme, the pause at the class II site delays the
appearance of significant amounts of run-off transcripts until ~80 s
after initiation of the reaction. However, in the reactions with
nicked, ThDel, and Y385A enzymes, run-off transcript is detected
20 s after initiation, and little or no pausing or termination at
the class II site is detected. Thus, bypass of the class II site by
these enzymes is not due to the fact that they pause but fail to
terminate. Instead these enzymes fail to both pause at the CAUCUGUU
element and to terminate at a class II terminator.

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Fig. 7.
Run-off transcription of
EcoRV-cut pDL68 (33) for the indicated times (s).
RNAP and template concentrations were as in Fig. 2. The sequence of the
pause/termination site in pDL68 is CAUCUGUU.
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Effects of RNAP Mutations or Nicking on Protection from RNase
T1--
Eight nucleotides of RNA are fully protected from RNase T1 in
a T7 RNAP EC, possibly because they are paired with the template (25,
38). An additional 4-6 nucleotides upstream of this strongly protected
region are partially protected from T1 digestion (25, 38). In an EC
formed with a nicked enzyme the strong protection of 8 nucleotides of
RNA is retained, but protection of the upstream region is reduced (38),
indicating that nicking of the N-terminal domain disrupts the
interaction between the RNAP and the upstream region of the RNA. To
determine whether the effects of the thumb mutations on EC stability
and termination are due to a similar change in the RNA-RNAP
interaction, we treated thumb mutant ECs with RNase T1. Fig.
8 shows representative data for the w.t.
complex (lanes 1-4), the nicked complex (lanes
5-8), and five thumb mutants (lanes 9-28). The
reduced protection of the upstream region of the RNA in the nicked
complex is apparent; T1 treatment of the nicked complex results in the
rapid degradation of the RNA to 8 nucleotides in length, whereas in the
reaction with the w.t. complex the 8-mer is generated more slowly and
11-15-mers accumulate. Quantitation reveals that the ThDel EC also
shows loss of protection of the upstream region of the RNA, but to a
more modest extent than the nicked enzyme. With the four point mutants,
the T1 digestion patterns are similar to that seen with the w.t.
complex, and all of the other thumb mutant complexes also showed
patterns of T1 sensitivity like the w.t. complex (not shown). Thus, the
reduced EC stability and termination for the thumb mutants cannot be
attributed to the kind of large change in the RNA-RNAP interaction that
is seen with the nicked enzyme.

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Fig. 8.
RNase T1 treatment of halted ECs. ECs
formed with linearized pPK10 and the indicated RNAPs were treated with
0.25 unit/µl of RNase T1 for the indicated times.
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DISCUSSION |
A Role for Helix N of the T7RNAP Thumb Subdomain in Stabilizing the
TC--
The effects of alanine substitutions at Arg391,
Ser393, and Arg394 indicate that these side
chains are involved in stabilizing the TC. Their contributions to
complex stability become important once the RNA reaches a length of
4-5 bases (Fig. 2). This supports modeling that places the
phosphate-ribose backbone of the RNA 4-5 bases away from the 3'-end
close to the 385-395 segment of the thumb (Fig. 1A). As
shown in Fig. 9 the different
contributions of the side chains within this segment to complex
stability can be interpreted in terms of their positions on helix N. The side chains of Ser393 and Arg394 face
toward the RNA and into the large template binding cleft of the
polymerase. The side chain of Arg391 faces away from the
cleft, but its
-carbon is on the same side of helix N as that of
Arg394, so Arg391 could swing into position to
interact with the RNA. The S393A and the R394A mutations destabilize
both ITCs (Fig. 2) and ECs (Fig. 5), whereas the R391A mutation
destabilizes ECs (Fig. 5) but does not markedly increase termination
during initial transcription (Fig. 2). Arg391 may only move
into position to interact with the RNA following the isomerization that
accompanies promoter release. Side chains 385-389, 392, and 395 either
point away from the RNA and template binding cleft or are too far from
the RNA, as indicated by modeling, to contact it (Fig. 9). Consistent
with this, alanine substitutions at these positions do not have large
effects on TC stability (Figs. 2, 3, and 5).

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Fig. 9.
Structure of a T7RNAP TC. The RNAP is
rotated by ~90° relative to Fig. 1, so that the long axis of helix
N points down. The template and nontemplate strands are cyan
and blue, respectively. The RNA is green, the
superimposed T7 DNA primer and ddNTP are red, and
nucleotides are numbered with respect to the 3'-nucleotide,
which is number 1. The amino acids mutated are shown in
space-filling representation. Residues Arg391,
Ser393, and Arg394 are in red;
Tyr385 and Asp388 are in cyan; and
Arg395, Lys392, Lys389,
Lys387, and Arg386 are in green. The
element deleted in the ThDel enzyme is magenta.
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Glycine Substitutions Are More Disruptive than Alanine
Substitutions--
Upon binding template, part of helix N that is
disordered in the apoenzyme assumes a helical conformation (3, 14, 15), indicating both that this part of the thumb subdomain is sensitively poised between helical and nonhelical conformations and that its secondary structure is important for function. To test this we introduced glycines at positions 391-394. Glycine is helix
destabilizing (36), so if the secondary structure of helix N is both
important for function and sensitive to perturbation, the glycine
substitutions should disrupt its activity. We found that glycine
substitutions had larger effects on termination during initial
transcription and on EC stability than did alanine substitutions (Figs.
2 and 5). In fact, although Lys392 points away from the RNA
and the template binding cleft and a K392A substitution does not affect
TC stability, a K392G substitution markedly destabilizes the EC and
increases termination during initial transcription. Similarly, although
an R391A substitution has little effect on initial transcription, an
R391G substitution has a large effect.
The Effects of the Mutations on EC Stability Are Influenced by
Template Topology--
A partial deletion of the thumb subdomain
results in an EC, which is exceptionally unstable on supercoiled, but
not linear, templates (Fig. 5). It has been suggested that this is
because an extended RNA-DNA hybrid forms on supercoiled templates and destabilizes the EC (17, 18). The point mutations in the thumb show a
DNA topology-dependent effect on EC stability similar to the thumb deletion. The effects of the thumb mutations on EC stability may be contrasted with the effect of nicking the N-terminal domain, which similarly destabilizes ECs on supercoiled or linear templates (Fig. 5 and Refs. 17 and 18). The decreased stability, processivity, and RNA displacement activity of the nicked EC has been proposed to be
due to disruption of an interaction between the single-stranded RNA and
an RNA binding site on the N-terminal domain (18, 23, 26, 31). The loss
of protection from RNase T1 in the RNA 8-14 bases away from the 3'-end
in the nicked EC supports this hypothesis, and the lack of an effect on
protection from RNase T1 with the thumb point mutants (Fig. 8)
reinforces the conclusion that the thumb and N-terminal domain
stabilize the TC through distinct mechanisms.
The Thumb Deletion and the Point Mutations Affect Initial
Transcription Differently--
Although the effects of the thumb
deletion and the point mutations on EC stability and RNA interactions
are similar (and distinct from the effects of nicking), the thumb
deletion and the point mutants have distinct effects on initial
transcription. Point mutations between 391 and 394 increase termination
for RNAs >4 bases in length, but the largest increases in termination
for the ThDel enzyme are for 8- and 9-base RNAs (Fig. 2). The small effect of the thumb deletion on termination for shorter RNAs is consistent with modeling (Fig. 9), which indicates that the amino acids
within the deleted segment are too far away to contact RNAs 4-6
nucleotides in length. The effect of the deletion on the stability of
TCs with longer (>7 bases) RNAs indicates either that the deleted portion of the thumb makes interactions with the RNA 8 or more bases
away from the 3'-end or that the destabilizing effect of the deletion
is only manifest once the RNA reaches a length of ~8 bases and the
RNAP releases the promoter.
NTP Binding Strongly Stabilizes the EC--
NTP binding reduces
the turnover rates of the w.t. and mutant ECs to below detectable
levels (Fig. 5). Similar observations were made by Mentesanas et
al. (18) and have also been observed in human immunodeficiency
virus, and type 1 reverse transcriptase (37). In addition to the strong
stabilization against dissociation, we have also found that NTP binding
restricts the lateral mobility (sliding) of the EC on the template
(38). EC stabilization caused by NTP binding may be due directly to
template-NTP-RNAP interactions, or it may reflect an NTP induced
isomerization from an "open" to a "closed" conformation in the
RNAP. Such an isomerization has been detected kinetically (39) and
proteolytically (40) in DNAPs homologous to T7RNAP and has been
observed directly in crystal structures (21, 41). The latter studies
revealed that this isomerization causes the fingers domain of the
polymerase to close around the template strand. Presumably, such an
NTP-induced isomerization could significantly enhance EC stability.
The Functional Importance of Different Side Chains between
Positions 385 and 395 Is Consistent with Their Conservation--
Three
amino acids (Tyr385, Arg391, and
Ser393) within 385-395 are well conserved among the phage
RNAPs, and at another two positions (Asp388 and
Arg394) there is conservation of charge (Fig.
1B). As judged from the effects of alanine substitutions,
Arg391, Ser393, and Arg394 make the
most important contributions to TC stability. To evaluate the
importance of charge at positions 391 and 394, we made lysine substitutions at both positions. Relative to the alanine substituted enzymes, both the R391K and R394K mutants form more stable ECs (Fig.
5), indicating that charge is a functionally important feature of the
side chains at these positions.
A Role for Helix N in Pausing/Termination at the Concatemer
Junction--
Mutation of the other two well conserved side chains
(Tyr385 and Asp388), which both project from
the outer surface of helix N (Figs. 1 and 9), does not affect TC
stability but does reduce pausing and termination at the T7 DNA CJ.
Proteolytic nicking of the N-terminal domain and deletion of part of
the thumb that is close to Tyr385 (Fig. 9) also reduce
pausing and termination at the CJ. The mechanism of termination at the
CJ is not well understood, so it is difficult to speculate on how these
alterations in RNAP structure affect response to the CJ. It is,
however, likely that nicking of the N-terminal domain and the thumb
mutations work through distinct mechanisms, because nicking leads to a
large change in protection from RNase digestion (38), as well as to
reduced EC stability (17, 18) and processivity (23, 26), and a defect
in RNA displacement (38). The Y385A and D388A mutations have none of these effects. The effect of nicking on CJ recognition may, therefore, be a consequence of a gross alteration in the RNA-RNAP interaction, whereas the effects of the Y385A and D388A mutations are limited to CJ
pausing and termination. It is possible that the Tyr385 and
Asp388 side chains directly recognize the CJ in the RNA. In
one model for the RNA path in the T7RNAP TC, the RNA passes through a
cleft formed by the thumb and N-terminal domain (4). The RNA, after emerging from this cleft, could contact
Tyr385/Asp388 on the outer surface of helix N
(Fig. 1). The distance between the RNA 3'-end and the RNA in contact
with Tyr385/Asp388 would then be ~9
nucleotides, which is consistent with the position at which termination
occurs relative to the CJ. However, a recent cross-linking analysis
proposes a distinct RNA path in which the RNA 8-10 nucleotides away
from the 3'-end lies near the RNAP promoter specificity loop (25). In
this case, a direct role for Tyr385 and Asp388
in recognition of the CJ appears unlikely, although it cannot be ruled
out because it is not known whether the RNA path in a normal EC differs
from that in a complex paused at the CJ. It is also possible that the
CJ is recognized, not through interactions with the RNA, but with the
DNA. Finally, it is possible that the effect of the Y385A and D388A
mutations is not due to disruption of RNA or DNA interactions but to an
effect on conformational changes important for responding to the CJ.
For example, because an isomerization in the thumb is important for TC
stability, it is possible that pausing or termination involves
conformational changes in the thumb that destabilize the TC. Mutations
in the thumb could interfere with these conformational changes.
Resolution of these questions requires a better understanding of the
RNA and DNA interactions in the T7RNAP EC and of the mechanism of pausing and termination at the CJ.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM 52522 (to R. S.), funds from the Robert H. Welch
Foundation, and a Fulbright-CONACYT fellowship (to L. G. B.).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: Dept. of Biochemistry,
University of Texas Health Sciences Center, 7703 Floyd Curl Dr., San
Antonio, TX 78284-7760. Tel.: 210-567-8782; Fax: 210-567-8778;
E-mail: sousa@biochem.uthscsa.edu.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M009866200
 |
ABBREVIATIONS |
The abbreviations used are:
RNAP, RNA
polymerase;
DNAP, DNA polymerase;
EC, elongation complex;
TC, transcription complex;
ITC, initial TC;
CJ, concatemer junction;
w.t., wild type;
TBE, Tris-Borate EDTA.
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