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INTRODUCTION |
The steps in transcription initiation performed by all
DNA-dependent RNA polymerases are promoter binding, DNA
strand melting, RNA chain initiation and nascent RNA chain formation,
and finally escape from the promoter sequences. Failure to escape from
the promoter results in the production of aborted RNA transcripts and
reiterative cycling through the open complex (1-4). Promoter escape
appears to occur concomitantly with the release of
70
factor following the synthesis of a specific length of RNA and the
conversion of the unstable initial transcribing complex to a stable
elongating complex (5-8). Depending on the promoter, abortive RNA
synthesis occurs to different degrees and can occur extensively enough
that it becomes rate-limiting for the synthesis of productive RNAs (2,
9).
Important determinants of abortive initiation are contained along the
region of melted DNA within the transcription complex encompassing the
non-transcribed and the initial transcribed sequences (ITS).1 Changes in the
composition of the ITS can alter the extent of productive RNA synthesis
(10) and cause dramatic variation in the total number of abortive
events and also alter abortive probabilities from position to position
within the ITS (11). Base residue changes specifically in the
non-template strand of the
10 promoter region, which weaken or
strengthen open complex stability, can lead to decreased or increased
abortive probabilities, respectively (12). Several examples of
regulation at the level of promoter escape have been reported. The
phage
29 promoter A2c was repressed by the phage regulatory protein
p4 through stabilization of RNA polymerase-promoter interactions to the
extent that promoter escape is reduced, and abortive transcription is
increased (13, 14). Alternatively, in phage P22, the Arc regulatory
protein has been shown to activate transcription by increasing promoter
escape through weakening the stability of open complexes (15). Upstream A-tracts, which activate promoter activity by improving complex formation between polymerase and promoter sequences (16, 17), can
conversely inhibit the activity of a promoter presumably by stabilizing
RNAP at the promoter to such a degree that it prevents efficient
promoter escape (18). In vivo, RNA polymerase bound at a
consensus promoter was shown to idle excessively along the ITS and
escape the promoter poorly (19). Thus, enhancement of polymerase-promoter interactions can activate or repress promoter activity depending on whether the rate-limiting step of transcription initiation is associated with polymerase binding, open complex formation (20), or promoter escape (21).
The determinants of promoter escape do not reside exclusively with the
promoter sequence but also involve components of the transcription
machinery, especially the
factor. Certain mutations in region 2.2 of
70 were found to affect promoter escape (22, 23). We
described previously (24, 25) the isolation of two mutant alleles of the gene encoding the
70 transcription initiation
factor, rpoD(P504L) and
rpoD(S506F), and characterized the in
vitro transcription behavior of these two mutant proteins,
70(P504L) and
70(S506F), reconstituted
into RNA polymerase holoenzyme. On three bacteriophage promoters, T7
A1, T5 N25, and T5 N25antiDSR, the mutant enzymes
transcribed to yield a reduced amount of abortive RNA chains compared
with the wild-type polymerase without increasing the abundance of
full-length RNA chains. These mutant enzymes behaved similarly at the
PR promoter (26), and it was determined that
70(P504L) and
70(S506F) RNA polymerases
do not accumulate the so-called "moribund" complexes, the
nonproductive complexes that yield only abortive products (27). Thus, a
lower tendency to form moribund complexes by these mutant polymerases
appears to bring about a lower production of abortive RNAs
predominantly (26). These results do not fulfill the expectation that a
lowered tendency to form non-productive complexes should increase the
probability of forming productive complexes, consequently resulting in
increased productive transcription.
To resolve this discrepancy, we extended the investigation of these
mutant polymerases to other well characterized highly abortive
promoters. As reported below, we found that on the E. coli gal P2 promoter, the mutant polymerases
drastically reduced abortive transcription while greatly stimulating
productive RNA synthesis when compared with the wild-type enzyme.
Subsequently, we determined the various transcription initiation
constants to pinpoint the basis of this altered transcription property.
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EXPERIMENTAL PROCEDURES |
Proteins--
The cloning of rpoD+,
rpoD(P504L), and
rpoD(S506F) and the overexpression and
purification of
70,
70(P504L), and
70(S506F) proteins have been described (25). Core RNA
polymerase was purchased from EpiCentre Technologies (Madison, WI).
Holoenzyme was reconstituted by mixing
70 with core RNA
polymerase at a ratio of 5:1 and incubating at room temperature for 15 min immediately prior to use in the transcription reactions.
Transcription--
The minicircle DNA template bearing the
gal P1 and P2 promoters was prepared as described
(28). Abortive initiation assays to measure the lag times for
-plot
analysis (29) were performed in potassium chloride buffer (40 mM Tris-HCl, pH 7.9; 10 mM magnesium chloride;
100 mM potassium chloride; 1 mM dithiothreitol;
100 µg/ml acetylated bovine serum albumin) with 2 nM of
template DNA per reaction and different concentrations of RNA
polymerase holoenzyme added to initiate transcription. The
gal P2-specific initiating dinucleotide ApU was present at
500 µM plus 10 µM
[
-32P]UTP (specific activity of 15 cpm per fmol).
Abortive initiation products were resolved on a 7 M urea,
20% polyacrylamide gel
(acrylamide:N,N'-methylenebisacrylamide = 59:1) (30) to
verify formation of the appropriate abortive products. Extensive
quantification at different polymerase concentrations was determined as
a percentage of the total radioactivity incorporated following
ascending paper chromatography as described (30). The radioactivity on
paper chromatograms was quantitated with the radioanalytic imaging
system from Ambis (San Diego, CA). The average time for open complex
formation,
obs, corresponds to the lag prior to the
final steady-state rate of product formation and was determined by
least square fit of the data to the theoretical exponential equation
derived by Hawley and McClure (31). Apparent binding and isomerization
constants (KB(app) and
kf(app)) were solved by least square fit
of the data to the classical derivation of McClure (29). Curve fitting
was accomplished with the SigmaPlot program (Jandel Scientific, San
Rafael, CA).
Steady-state transcription reactions were performed in potassium
glutamate buffer (20 mM Tris acetate, pH 7.8; 10 mM magnesium acetate; 100 mM potassium
glutamate; 10 mM
-mercaptoethanol) with 10, 20, 50, or
100 µM [
-32P]ATP (specific activity of
50 cpm per fmol), GTP, CTP, and unvarying UTP (at 10 µM)
for 10 min at 37 °C in a reaction volume of 20 µl. Reactions were
initiated by mixing 50 nM RNA polymerase holoenzyme together with 2 nM template plus nucleoside triphosphates
all prewarmed to 37 °C for 2 min.
Single-round transcription kinetics (Fig. 7) were performed by mixing
RNAP (20 nM) with template (2 nM) followed by
preincubation for 5 min prior to the simultaneous addition of heparin
(25 µg/ml) with 100 µM ATP, GTP, CTP, and 10 µM [
-32P]UTP (specific activity of 50 cpm per fmol) in a reaction volume of 100 µl, and 10-µl aliquots
were removed at subsequent times. Reactions were stopped, precipitated,
resuspended, and treated as described previously (25). Abortive RNAs
were analyzed by electrophoresis of 1/4 of each sample on a 7 M urea, 25% polyacrylamide gel
(acrylamide:N,N'-methylenebisacrylamide = 12.5:1).
Electrophoresis and buffer conditions were as described before (25).
Full-length RNAs were separated on a 7 M urea, 6%
polyacrylamide gel (19:1) using a conventional Tris borate-EDTA buffer
(TBE) system. Radioactivity was quantified using a Storm PhosphorImager
and ImageQuant program version 4.2 from Amersham Biosciences.
Determination of Open Complex Stability--
An excess of RNA
polymerase holoenzyme (100 nM) was mixed with template (2 nM) in potassium chloride buffer (above) in a volume of 100 µl and incubated at 37 °C for 10 min to allow the formation of
open complexes. Heparin or poly[d(AT)] was then added to RNA polymerase-template mix to a final concentration of 25 µg/ml. At
subsequent times aliquots of 10 µl were removed and mixed with an
equal volume of a pre-warmed chase mixture (400 µM ATP,
GTP, CTP; 10 µM [
-32P]UTP (200 cpm per
fmol); 25 µg/ml sodium poly[d(AT)] or sodium heparin).
Transcription reactions were carried out for an additional 5 min at
37 °C and then stopped by mixing with 5 µl of 70% formamide, 100 mM EDTA, 1× TBE, 0.025% bromphenol blue, and 0.025%
xylene cyanol. Full-length RNAs were analyzed and quantified as
described above. An initial or zero time point was taken prior to the
addition of heparin or poly[d(AT)].
Gre Factor Purification--
GreA and GreB were purified from
the Escherichia coli strain JM109 bearing the GreA and GreB
overexpression plasmids, pDNL278 and pGF296, respectively, as described
(32).
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RESULTS |
70 Mutations--
Missense alleles
rpoD(P504L) and rpoD(S506F)
contain single amino acid substitutions of leucine for proline and
phenylalanine for serine at residues 504 and 506, respectively, in
subregion 3.2 of
70 (Fig.
1); subregion assignment within region 3 is based on the structural domains identified in the Thermus
thermophilus RNAP holoenzyme crystal structure of Vassylyev
et al. (41) rather than the original assignment based on
sequence alignment (44). Proline 504 and serine 506 are 2 of only 10 residues in region 3 that are invariant among the primary
factors
(Fig. 1).

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Fig. 1.
Mutational changes in region 3.1. Four
protein regions and subdivisions highly conserved among the major factors are represented on a linear map of the 613-amino acid E. coli 70 (44). Region 3 lies between conserved
regions 2 and 4 involved in the recognition and binding of the 10 and
35 promoter elements, respectively. The amino acids of region 3 are
shown above the 70 map; residues of identity
among all major factors are denoted by large boldface
letters. Missense substitutions at amino acid positions 504 and
506 are indicated.
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In Vitro Transcription System--
The gal P1 and P2
promoters have been extensively characterized in vitro with
respect to RNA polymerase binding, rate of open complex formation,
abortive RNA synthesis, and promoter escape (28, 30, 33). The activity
of gal P1 is limited by promoter binding (33), whereas that
of gal P2 is intrinsically limited at the step of promoter
escape (28). The gal P1 promoter lacks an identifiable
35
promoter element; however, polymerase binding is aided by an extended
10 promoter element (34). The gal P2 promoter has both the
35 and
10 elements with a non-optimal spacing of 16 bp in addition
to an extended
10 promoter element (35) (Fig.
2). Following binding at gal
P2 there is extensive "idling" or abortive cycling of polymerase
along the initial transcribed sequences prior to promoter escape. The
probability that an RNA chain initiated at P2 is aborted rather than
extended is estimated to be greater than 95%. For reasons that are
unclear, abortive yields are especially high at specific sites,
i.e. at positions +2, +3, and +6, within the initial
transcribed sequence of gal P2 (28) (Fig.
3). In addition, at high UTP
concentrations polymerase tends to "stutter" at the run of three U
residues encoded in the initial sequence ATTTC of gal P2
after initiating at the normal A residue start site (36). Stuttering
refers to the nonproductive mode of reiterative incorporation of UMP
producing poly(U) RNA chains that are released at a variety of lengths
and can grow to hundreds of nucleotide residues (37). At low UTP
concentrations, this stuttering does not occur. This substrate
dependence is one feature that distinguishes stuttering from the normal
abortive RNA synthesis; the latter occurs readily at low and high
nucleotide concentrations (11).

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Fig. 2.
Promoter region of mini-gal
P1 and P2. The RNA synthesis start site of gal P2
is located 5 bp upstream of the gal P1 start site. The
Pribnow boxes for gal P1 and P2 overlap and are indicated by
boxes labeled 10, the shaded box is for
gal P1. The overlines indicate extended 10
sequences, TG, for both gal P1 and P2. The 35
promoter element for gal P2 is labeled and indicated by the
box. The positions within the initial transcribed sequences
of gal P2 where the majority of "natural" abortive
transcriptions occur are labeled, denoted by large boldface
letters and asterisks. Transcripts initiating at
gal P2 terminate on the mini-gal template at the
rpoC terminator 125 bp downstream and those from
gal P1 terminate 120 bp downstream as indicated (28).
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Fig. 3.
Complete RNA synthesis reactions
on gal P1 and P2. A, the production of
naturally abortive RNAs and full-length terminated RNAs arising from
the mini-gal template was visualized. Transcription was
performed as described under "Experimental Procedures" with
increasing concentrations of ATP, GTP, and CTP as indicated with UTP
held constant at 10 µM. Abortive RNA species were
separated on a 25% polyacrylamide gel and full-length RNAs on a 6%
polyacrylamide gel (see "Experimental Procedures"). RNA was labeled
with [ -32P]ATP. Length of RNAs are labeled as follows:
2, dimer; 3, trimer; 4, tetramer; etc.
(P1)120 = gal P1 and (P2)125 = gal P2 transcripts. B, PhosphorImager counts of
the radioactive abortive and full-length RNAs at different nucleoside
triphosphate concentrations determined from the gel of A. Wild-type RNAP, filled circles; 70(P504L)
RNAP, open triangles; and 70(S506F) RNAP,
open circles. C, the ITS of gal P1 and
P2. The relative degree of reduction in the abundance of abortive
transcripts produced by 70(P504L) RNAP at different
positions along the ITS of gal P2 is indicated by the length
of downward arrows.
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The recombination-excision system of Choy and Adhya (28) was used to
generate a supercoiled minicircle template containing only the
gal P1 and P2 promoters upstream of a single Rho-independent terminator. This "mini-gal" template specifies the
transcription of short abortive RNAs as well as a 120- and
125-nucleotide RNA originating from gal P1 and P2,
respectively, without contaminating RNAs arising from endogenous
promoters, a limitation usually encountered when using regular
supercoiled plasmids as templates.
Synthesis of Abortive and Full-length RNAs--
Multiple round
in vitro transcription reactions were performed in the
presence of all four NTPs to determine the continuous production of
abortive and productive gal RNAs (11). We used increasing
concentrations (10-100 µM) of ATP, GTP, and CTP and a
fixed concentration of UTP (10 µM); the UTP concentration
was kept low to prevent polymerase stuttering (see above). Because both
gal P1 and P2 initiate with an A residue, the transcripts were all 5'-labeled at a constant specific activity with
[
-32P]ATP. End labeling of RNA transcripts has the
advantage of allowing direct stoichiometric comparison of radioactivity
incorporated into different RNA species regardless of size. The
wild-type RNA polymerase transcribes gal P1/P2 to yield an
abortive RNA ladder up to a length of 7 nucleotides and the terminated
P1 and P2 RNA of 120 and 125 nucleotides, respectively (Fig.
3A). The abortive RNAs arise predominantly from
gal P2 because very little difference in abortive products
was observed from a mini-circle template bearing a gal P1
knock-out mutation (28). With the wild-type enzyme, the level of
abortive and productive RNA synthesis increased concomitantly with
increasing NTP concentration, suggesting that the abortive pattern did
not result from NTP substrate limitation. Rather, a higher
concentration of NTP supported a higher frequency of initiation, with a
fraction of the initiated chains becoming productive RNA and the
remainder abortive.
Compared with the wild-type enzyme, mutant polymerases accumulated many
fewer abortive RNA chains and more full-length RNA chains from both
gal promoters (Fig. 3A). The reduction of
abortive RNA chains appears to be differentially affected by the mutant polymerases. The dimer RNA is reduced about 40%, and the trimer and
hexamer RNAs are reduced by 4- and 10-fold, respectively (Fig. 3B). Relative reduction of abortive RNA by the mutant RNA
polymerases at all positions along the initial transcribed sequence of
gal P2 is indicated with downward arrows in Fig.
3C.
The mutant RNA polymerases also gave rise to increased accumulation of
both gal P1 and P2 transcripts. The abundance of full-length RNA chains produced by mutant enzymes is higher by about 2-fold for
gal P1, 4-fold for gal P2, and 3-fold overall
(Fig. 3B). Several interesting comparisons can be made on
the changes in productive synthesis. On a number of promoters tested
previously, the
70 mutant polymerases showed reduced
abortive transcription but no increase of productive synthesis (25,
26); thus, increased productive synthesis, which can be equated with an
enhanced ability at promoter escape, by the mutant enzymes appears to
be a promoter-dependent phenomenon. We further noticed that
at low NTP concentrations with the wild-type enzyme, P1 was the
predominant full-length transcript, but at higher NTP concentrations,
P1 and P2 RNA achieved parity. This observation can be explained by the
excessive idling of wild-type polymerase at the P2 promoter relative to
P1 (28), and the extent of idling was reduced when NTP concentration
was raised. For the mutant enzymes, however, P2 was the more abundant productive RNA at all NTP concentrations. Thus, the
70
mutations seemed to have brought about two effects on gal P2 transcription, overcoming an NTP concentration-dependent
ability at escape and increasing the intrinsic rate of promoter escape.
To compare further the abortive and productive synthesis by the
wild-type and mutant enzymes, we plotted the abortive and productive
yields (see Ref. 11; i.e. the mole fraction of abortive and
full-length RNA per total product) and abortive probabilities at each
position along the initial transcribed sequence of gal P2
(Fig. 4). This revealed aspects of mutant
enzyme behavior not obvious from the data in Fig. 3. The abortive yield
(Fig. 4A) of dimer RNA is actually greater for the mutant
polymerases, that of trimer and hexamer RNA is significantly reduced,
and other abortive RNAs are represented by about equal yields.
Full-length gal P2 RNA is increased from 1% of total RNA
for the wild-type enzyme to ~6 and 5% for P504L and S506F
70 containing holoenzyme, respectively (Fig.
4A). Abortive probabilities were determined at each position
by correcting the percentage of each abortive RNA chain by the fraction
of polymerases that actually advanced to that position. This revealed
that the wild-type and mutant enzymes had very similar overall
probabilities of producing abortive RNAs of 5 nucleotides or shorter.
Most pronounced differences occurred at +6 and +7 where the
probabilities of forming the 6- and 7-mer abortive transcripts were
reduced 50-70% for the mutant enzymes (Fig. 4B). Perhaps
not coincidentally, these positions correspond to the maximum length of
RNA synthesized prior to escape and thus likely demarcate the positions
where the initial transcribing complex makes the transition to an
elongating complex. Taken together, the above analysis allows us to
conclude that the mutant polymerases are more facile at promoter
escape, thus producing fewer abortive RNA and more full-length RNA.

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Fig. 4.
Abortive yields and probabilities on
gal P2. A, the percent of total
incorporated counts represented by each length of RNA species, called
the abortive yield, is presented (11). For this analysis RNA products
transcribed with 50 µM [ -32P]ATP, GTP,
CTP, and 10 µM UTP were quantified. This was the highest
concentration of nucleoside triphosphates for which dimer, trimer, and
unincorporated radioactive ATP were sufficiently well resolved so that
the signal to noise ratio for accurate quantitation of these small RNA
chains was tolerable. Wild-type RNAP, black bar;
70(P504L) RNAP, lightly shaded bar;
70(S506F) RNAP, darkly shaded bar.
B, the abortive probabilities were calculated by
Equation 1 (see Ref. 11),
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(Eq. 1)
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where Pi is the abortive probability
that the transcript will be aborted from the initial transcribed
complex at the ith position. Xi is the
abortive yield of the ith RNA species (in percent).
Histogram bars are the same as in A. Total
PhosphorImager counts were 2.02 × 106 for wild-type
enzyme, 1.43 × 106 for P504L enzyme, and 0.98 × 106 for the S506F enzyme.
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Open Complex Formation at gal P2--
To gain insight into the
differential transcriptional outcome during RNA synthesis from
gal P2 by wild-type and
70 mutant enzymes, we
determined and compared the various transcription initiation constants
for these enzymes. First, we evaluated whether the
70
mutants could affect the general process of promoter binding or open
complex formation by determining the apparent equilibrium binding
constant, KB, and the forward rate constant, kf, of open complex formation at the gal
P2 promoter. This was done by the steady-state abortive initiation
assay using a subset of nucleoside triphosphates followed by the
derivation of
plots (29, 30). Because the binding of polymerase at gal P1 and P2 is mutually exclusive, we specifically
determined the apparent KB and kf
values (KB(app) and
kf(app)) only for P2 by using the
initiating dinucleotide ApU and [
-32P]UTP. These
limited substrates permit the synthesis of only the trimer (pApUpU) and
tetramer (pApUpUpU) RNAs, the production of which was verified by PAGE
(Fig. 5A). By monitoring the
length of time required for the appearance of RNA following the
addition of enzyme to template and substrates, the average lag time
(
obs) for polymerase binding and open complex formation
was measured at five different enzyme concentrations to determine
KB and kf values for the
wild-type and mutant enzymes. At the low polymerase concentrations the
steady-state rate of trimer and tetramer RNA production was lower for
the P504L mutant than the wild-type enzyme (Fig. 5B).
However,
obs was approximately the same at all RNA
polymerase concentrations (Fig. 5C). Similarly for the S506F
mutant, limited measurements at low and high polymerase concentrations
revealed approximately the same
obs value (data not
shown). From
obs, we obtained the values for
KB(app) and
kf(app) according to Goodrich and
McClure (30); these values are 10.5 ± 1.2 × 106
M
1 and 9.76 ± 0.4 × 10
2 s
1 for both the wild-type and the
mutant enzymes. Thus, the mutant polymerases appear to bind promoter
and form open complexes normally on gal P2.

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Fig. 5.
Determination of open complex formation
constants on gal P2. A, kinetics of
accumulation of gal P2-specific ApUpU and ApUpUpU abortive
products. Shown are sample time points from 0.5 to 10 min of reactions
initiated by addition of 100 nM of either wild-type or
70(P504L) bearing RNA polymerase holoenzyme to 2 nM mini-gal template in the presence of ApU and
[ -32P]UTP. Radioactive reaction components were
fractionated on a polyacrylamide gel system that separates short RNAs
based on charge rather than size ("Experimental Procedures") (45).
B, kinetics of percent radioactivity incorporated using 2 nM mini-gal template and 100 nM or
10 nM RNAP. Percent incorporation into trimer and tetramer
RNA was determined following paper chromatography as described under
"Experimental Procedures." C, observed lag times
( obs) were plotted as a function of the reciprocal of
RNAP concentration. obs were determined at five RNAP
concentrations from kinetics of percent radioactive incorporation
determined as described under "Experimental Procedures." Wild-type
RNAP, filled circles; 70(P504L) RNAP,
open circles.
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Stability of RNA Polymerase-Promoter Open Complexes--
A
correlation between lower open complex stability and increased rates of
productive RNA synthesis was originally noted for mutant lac
promoter variants (9). In its simplest interpretation, this correlation
suggests the notion that the formation of a stable open complex and
promoter escape are antagonistic processes. We next examined the
stability of pre-formed open complexes of wild type, and mutant
70 polymerases on gal P1 and P2 were
monitored in the presence of a nonspecific competitor, heparin or
poly[d(AT)]. Open complexes were formed in the absence of NTP using a
50-fold molar excess of RNA polymerase to template to maximize promoter
occupancy. Heparin was added next, and the promoter occupancy of
gal P1 or P2 was assayed at subsequent times by adding high
concentrations of NTP and monitoring the production of full-length RNA.
The wild-type enzyme formed very stable open complexes on the
gal promoters as found previously (33). After 2 h, only
about 5-20% of the wild-type open complexes had dissociated from
either gal P1 or P2 (Fig. 6).
By contrast, the mutant polymerases exhibited a 50% loss of the
gal P1 and gal P2 open complexes at ~80 and 40 min, respectively, after the addition of heparin (Fig. 6). The shorter open complex half-life with the mutant polymerases is not caused by the
competitive nature of heparin (38); we compared the effect of heparin
and poly[d(AT)] as nonspecific competitors and found that they gave
comparable results (data not shown). The open complex half-life for the
mutant polymerases corresponds to an
k
f(app), value of 2.1 and 0.4 × 10
4 s
1, respectively, for gal P1
and gal P2.

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Fig. 6.
Stability of open complexes formed on
gal promoters. A 100-fold molar excess of RNA
polymerase was incubated with mini-gal template for 10 min
at 37 °C followed by addition of either heparin or poly[d(AT)] to
a final concentration of 25 µg/ml. At subsequent times aliquots were
removed and chased with nucleoside triphosphates to monitor
transcription activities at gal P1 and P2 (see
"Experimental Procedures"). Activities are expressed as a fraction
of the activity prior to addition of competitor and represent the
fraction of open complexes remaining at the indicated times. Panels
labeled P1 and P2 indicate promoter occupancy at
gal P1 and gal P2, respectively. RNAP,
filled circles; 70(P504L) RNAP, open
triangles; 70(S506F) RNAP, open circles.
Zero time point values for PhosphorImager counts of gal P1
and P2 transcripts in the presence of heparin for wild-type enzyme were
2.40 × 105 and 2.72 × 105; for
P504L enzyme, 2.71 × 105 and 2.74 × 105; and for S506F enzyme, 2.72 × 105 and
2.98 × 105, respectively.
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Comparing the Kinetics of Promoter Escape and Abortive
Transcription--
So far, we have shown that the mutant polymerases
are not compromised at open complex formation at gal P2, but
the open complexes formed are less stable than that of the wild-type
enzyme. When NTP is present, the mutant polymerases are also more
facile at promoter escape. To confirm that the
70
mutations have altered the rate of escape, we assayed the lag time for
the appearance of each RNA species under single-round conditions. The
lag time was determined for the 3-mer, 6-mer, gal P1, and
gal P2 RNAs by quantifying these RNA species following the
preincubation of an excess of polymerase to mini-gal
templates followed by heparin and all four nucleoside triphosphates
(see "Experimental Procedures"). As shown in Fig.
7A (right-hand
column), the lag time for the appearance of the 3-mer and 6-mer
RNAs is about 10-15 s for both the mutant and wild-type polymerases.
This value closely matches the rate of open complex formation
determined by
-plot analysis at this polymerase concentration (see
Fig. 5B; 100 nM RNAP). Similarly, the lag time
for the appearance of the gal P1 transcript, also 10-15 s,
is virtually identical for all enzymes. A major difference, however, is
observed for the time required for the appearance of the gal
P2 transcript; the wild-type polymerase requires about 45 s, while
both mutants require 10-15 s. Under these conditions only about 10-15
s are required to achieve the first initial transcribing complex (ITC);
thus, unlike the wild-type enzyme, no detectable idling of mutant
polymerases occurs at gal P2. These results indicate that
the mutant enzymes differ from the wild-type enzyme in that they appear
to escape the gal P2 promoter without idling.

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Fig. 7.
Kinetics of RNA synthesis from gal
promoters. Kinetics of the accumulation of hexamer
(6-mer), trimer (3-mer), full-length
gal P1 (P1), and gal P2
(P2) transcripts were measured in the presence of 50 µM ATP, GTP, CTP, and 10 µM
[ -32P]UTP. Transcription conditions, resolution, and
quantitation of RNA species were as in Fig. 4, except that heparin (50 µg/ml) was added simultaneously with nucleoside triphosphates.
Wild-type RNAP, filled circles; 70(P504L)
RNAP, open triangles; 70(S506F) RNAP,
open circles. A shows the kinetics of RNA
accumulation; the left-hand column represents a full time
scale of 15 min; the right-hand column represents a time
scale of 2.5 min. B shows the ratio of hexamer RNA to
gal P2 full-length RNA (6-mer/gal P2) and trimer
to gal P2 full-length RNA as a function of time after
substrate addition.
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Furthermore, because formation of the 6-mer RNA occurs with no
detectable further lag, and the 6th and 7th positions in the initial
transcribed sequence appear to demarcate the transition point of the
ITC to the elongation phase, the slow step preventing the wild-type
polymerase from escape may be the escape reaction itself that most
likely occurs just after position +6. One straightforward interpretation of these results is that the mutant
70
proteins are rapidly released from the ITC compared with the wild-type
enzyme at a critical RNA length following 6 nucleotides, thus allowing
rapid promoter escape.
Formation of Moribund Complexes by gal P2--
Previously, the
PR promoter was shown to have a high tendency
to form the moribund abortive complexes with wild-type
polymerase (27), but that tendency was lowered with the
70 P504L and S506F mutants (26). On the gal
promoters, the wild-type polymerase showed a similar high propensity to
form moribund complexes; this is evidenced by the continuous
accumulation of abortive RNA transcript which increased linearly for 15 min of transcription while the full-length transcripts accumulated
linearly for less than 4 min and then began to plateau (Fig.
7A, left-hand column). As was observed before
(26, 27), this differential pattern of RNA accumulation is indicative
of the existence of two populations of initial transcribing complexes:
one productive and the other moribund. In this single-cycle
transcription, the productive complexes are rapidly depleted following
promoter escape, but the long lived moribund complexes are trapped at
abortive cycling to produce the short abortive RNAs continuously.
Interestingly, the moribund fraction of gal P2 complexes
formed with mutant polymerases is drastically reduced. This can be seen
when comparing the rate of abortive RNA synthesis of mutant enzymes
with the wild-type enzymes (Fig. 7A, 3-mer and
6-mer); however, this comparison assumes that the chain
initiation and abortive cycling rates of the wild-type and mutant
moribund complexes are the same.
The existence of moribund complexes is even more clearly demonstrated
when one plots the ratio of abortive to productive RNAs over the time
course of the transcription reaction (Fig. 7B). For the
wild-type enzyme, the initial ratio of 6-mer and 3-mer abortive RNAs to
productive transcripts is 8 and 15, respectively. These levels stay
fairly constant between 3 and 5 min after the start of the reaction
when the abortive to productive ratio begins to rise sharply, reaching
a value of 20 and 45, respectively. The initial constant abortive to
productive ratio is the result of parallel increases of abortive and
productive RNA at early times; the later sharp rise results when
productive synthesis has ceased and the abortive synthesis by the
moribund complexes continues. For the
70 mutant
holoenzymes, the initial ratio of abortive to full-length RNAs is close
to unity at the beginning of the reaction and increases only modestly,
up to 8-fold at the most, at later times. These patterns of abortive
versus productive synthesis indicate that the mutant
polymerases form a much lower percentage of moribund complexes at
gal P2, in agreement with the observation made with the
PR promoter (26).
Effect of Gre Factor Addition on Abortive
Transcription--
Earlier, GreA and GreB transcript cleavage factors
were shown to stimulate promoter escape in an escape-limited promoter
(32). Recently, these factors were found to relieve the initiation
arrest of moribund complexes (39). The above results suggesting that the highly abortive nature of gal P2 on wild-type RNA
polymerase can be attributed in part to its high tendency to form
moribund complexes prompted us to test the sensitivity of
gal P2 abortive transcription to the Gre factors. As shown
in Fig. 8, the presence of GreA and GreB
factors dramatically reduced the formation of the 6-mer and 7-mer
abortive RNAs but showed almost no effect on shorter (<6-mer) abortive
RNAs. At the same time, a 3-5-fold increase in full-length
gal transcripts is evident. This is consistent with the
observed effects of Gre factors on the moribund complexes at the
PR promoter (39). Thus, the Gre factors also
stimulate the conversion of gal moribund complexes into
productive complexes. Addition of excess Gre factors to the mutant RNAP
holoenzymes had no observable effect on gal P2 transcription
apart from those previously attributed to the presence of the mutant
factors per se (data not shown).

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Fig. 8.
Gre factor sensitivity of gal
P2 promoter transcription. Purified GreA and GreB were added
at an increasing stoichiometry to RNA polymerase (Gre/RNAP)
as indicated starting at a ratio of 1:1 stoichiometry and raising the
Gre factor stoichiometry as high as 10:1. Abortive and productive
full-length RNAs were visualized as described in the legend to Fig. 3.
Length of RNA species is indicated. Transcription was performed with 50 µM ATP, GTP, CTP, and 10 µM UTP. RNA was
labeled with [ -32P]ATP, as described under
"Experimental Procedures."
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DISCUSSION |
Differential Behavior of
Mutants on Different
Promoters--
In this report, we investigated the transcription
properties of RNA polymerase holoenzyme containing either
70(P504L) or
70(S506F) on the highly
abortive cellular promoter gal P2 and found that the mutant
polymerases reduced abortive RNA synthesis and increased the abundance
of full-length RNA. By examining the various rate constants associated
with transcription initiation, we showed that the mutant enzymes are
equally capable as the wild-type enzyme in binding and melting the
promoter DNA to form open complexes. However, the open complexes with
the mutant enzymes are less stable, exhibiting a much lower open
complex half-life than the wild-type enzyme. This difference alone
between the mutant and wild-type enzymes can account for the diminished
level of abortive RNA synthesis by the mutant enzymes, basically
reflecting a lower amount of "trapped" open complexes at the
promoter. In this regard, gal P2 transcription by the mutant
enzymes resembles
PR transcription in that the amount of
open complex formed is less than that with the wild-type enzyme (26).
This explanation may also account for the reduced abortive
transcription from T7 A1, T5 N25, and T5 N25antiDSR
promoters by the mutant enzymes, although in that study, the stability
of the open complexes was not examined (25).
More interestingly, the
70 region 3.2 mutant polymerases
showed differential effect with regard to increasing promoter escape. These mutant enzymes did not stimulate promoter escape from T7 A1, T5
N25, T5 N25antiDSR, nor
PR but greatly
enhanced productive synthesis from gal P2. On gal
P2, the enhanced productive synthesis appears to have resulted from two
factors as follows: one, a greatly diminished fraction of moribund
complexes (Fig. 7A, top frames) and therefore a
greatly elevated fraction of productive complexes that carries out
full-length RNA synthesis; two, the mutant enzymes display an increased
rate of promoter escape (i.e. a shorter
t1/2 of productive synthesis of ~2 min) compared
with that of the wild-type enzyme (t1/2 of ~5 min;
see Fig. 7A, bottom frame). The increase in
productive synthesis was not observed with
PR, although
the mutant enzymes were shown to form a lower fraction of moribund
complexes at this promoter (26). What might be the cause of this
discrepancy? Is the stimulation of promoter escape by the mutant enzyme
simply a promoter-dependent phenomenon? This would be
likely if the promoters are very different in sequence, but this does
not appear to be the case. Is it possible that the number of abortive
complexes can be lowered without necessarily increasing the number of
productive complexes? Another possible difference between our previous
and present results could be the source of RNA polymerase; however, our
previous and current results were obtained using identical batches of
commercial
-free core RNAP (obtained from Epicentre Technologies,
Madison, WI) for holoenzyme reconstitution (see "Experimental
Procedures" and Ref. 25). An important factor that likely accounts
for the above discrepancy is the supercoiling status of the promoter.
Here, gal P2 transcription was performed with supercoiled
minicircle templates, whereas those of
PR, T7 A1, T5
N25, and T5 N25antiDSR were all performed with linear
templates. That supercoiling of the template can lead to a greatly
increased level of productive synthesis is currently under
investigation and will be reported elsewhere. We summarize the
initiation constants measured for gal P1 and gal
P2 in Table I. We further use a summary
diagram to illustrate the steps of transcription initiation where the
70 mutations have brought about different effects for
the wild-type versus the mutant enzymes (Fig.
9).

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Fig. 9.
Kinetic scheme of wild-type and mutant enzyme
behavior on gal P2 promoter. The symbols
represent the following: R, wild-type RNAP holoenzyme;
R', mutant 70 RNAP holoenzyme; P,
promoter; RPc, closed promoter complex;
RPo, open promoter complex; ITC, initial
transcribing complex; ITCp, productive ITC;
ITCu, unproductive ITC; EC, elongation
complex. KB, equilibrium binding constant;
kf, forward rate of open complex formation;
k f, rate of decay of open complex. The
thickness of the arrow is meant to indicate the
different extent of a reaction.
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Based on the current findings, we propose that the P504L and S506F
mutations lead to a rapid release of
70 from the initial
transcribing complexes to allow RNA synthesis to proceed to longer
lengths. This idea is consistent with the original proposal of Hansen
and McClure (5) that the presence of
70 sterically
hinders the elongation of the nascent RNA beyond a specific length and
that the release of the
factor is a physical prerequisite for
continued RNA synthesis. Specifically, the idea of a physical
competition between
70 region 3 and the nascent RNA in
the "RNA exit channel" is verified by analysis of the x-ray
crystallographic structures of prokaryotic RNA polymerase holoenzyme
recently reported (Refs. 40 and 41 and see below). In these two
studies, it is predicted in the former and observed in the latter that
70 region 3.2 occupies the entire length of the RNA exit
channel and is predicted to cause steric clash with the advancing
nascent RNA transcript during initial steps of RNA polymerization,
providing the physical basis for abortive transcription.
Finally, consistent with the observation that moribund complexes are
sensitive to the presence of the Gre transcription factors and fail to
accumulate in their presence (39), we tested and observed a reduction
in abortive RNA synthesis at the gal P2 promoter concomitant
with a stimulation of full-length productive RNAs in the presence of
Gre factors (Fig. 8). In their study, Shimamoto and colleagues (39)
concluded that Gre factors cause a direct conversion of moribund
complexes into productive complexes by opening a pathway of
inter-convertibility between these two types of initial transcribing
complexes. We propose that the
mutant proteins also fail to
accumulate moribund complexes because they form mutually exclusive
productive complexes (Fig. 7B). The high propensity to form
productive complexes is due to structural alterations in the holoenzyme
in which the region 3.2 mutant
factors do not sterically hinder the
positioning of the nascent RNA in the RNA exit channel as tenaciously
as wild-type
70. Furthermore, we observed no additive effect of
excess Gre factor on mutant RNAP behavior likely indicating that mutant
factors mimic the presence of the Gre factors, namely allowing the
interconvertibility of moribund into productive initial transcribing complexes.
Phenotypic Suppression of the ppGpp0
Condition--
The two
70 mutants, P504L and S506F,
characterized in this study were originally isolated as phenotypic
suppressors of strains devoid of the transcriptional effector ppGpp.
Strains deleted for the genes encoding the two known ppGpp synthetases
in E. coli, the relA and spoT genes,
lose the ability to accumulate the intracellular stress messenger ppGpp
and as a consequence display pleiotropic defects in growth and stress
adaptation (42). A newly discovered role of ppGpp in the regulation of
factor competition (43) sheds new light on the behavior of these
mutants with respect to their ability to suppress defects
associated with the intracellular loss of ppGpp. It was originally
reported that a striking characteristics of these two
mutants was
their lower affinity for core RNA polymerase leading to a lower
abundance of
70 holoenzyme in the cell (24). In light of
the newly understood role of ppGpp with respect to lowering
70 core affinity, we can now interpret the lowered
ability of these
mutants to associate with core RNAP as mimicking
the normal effect of ppGpp in vivo. Furthermore, it seems
that effects leading to increased alternative
competition are
fundamental to the global effect of ppGpp.
Structural Basis and Mechanism of Mutant
70
Behavior--
In the crystal structure of the open complex (40),
domains 2 and 4 are located on the surface of the holoenzyme
interacting with base residues of the
10 and
35 promoter elements,
respectively. The predicted path of the
region 3 peptide that links
domains 2 and 4 (referred to as the "Linker Domain") first descends
into the enzyme crevice toward the active site and then makes a
"hairpin turn" away from the active site to lay down the remainder
of acidic region 3.2 into the path of the basic RNA exit channel. The
presence of region 3.2 in the RNA exit channel is conjectured to pose
steric clash between the advancing nascent RNA and the
70 protein,
causing abortive release of the RNA (40, 41).
Residues 504 and 506 of
70 are located in the beginning
stretch of region 3.2 according to the structural domain assignments of
Vassylyev et al. (41) and may directly alter the path of the
peptide backbone in the linker domain. Overall the
subunit appears to be composed entirely of
-helices connected by either turns or loops (41), and the two
70 mutations we
examined are located, by structural analogy, at the end of the 11th
-helix in the T. thermophilus holoenzyme structure (41).
By using the SOPMA secondary structure prediction algorithm, both
mutations are predicted to extend the
-helical potential of the 11th
helix beyond proline 504 to position 510 where the next proline residue
occurs. This would extend the 11th
-helix through the N-terminal
stem of the hairpin sequence ending just prior to the turn in the
hairpin turn structure as discussed above and would re-direct the
70 peptide backbone away from the catalytic center and
away from the RNA exit channel. By disrupting the normal helical and
hairpin turn pattern, P504L and S506F mutations lead to a situation
where
3.2 is now likely positioned loosely in the RNA exit channel. Consequently,
3.2 becomes less of an impediment to the advancing RNA, lowering abortive initiation; at the same time, it might be more
easily displaced from the RNA exit channel, facilitating
release
and resulting in promoter escape. Consistent with this notion is the
observation that mutant
70 proteins preferentially reduce abortive
probabilities at RNA lengths of 6 and 7 and not 2-5 nucleotides (Fig.
4B). Thus, the abortive probabilities are reduced
specifically at a length when
release is likely occurring (Fig. 4).
In addition, the supposition that the P504L and S506F mutations in
70 alter and disrupt the normal path of region 3.2 with
respect to its normal disposition within the RNA exit channel is
supported by the physical defects displayed by these mutant
70s. The loss or "loosening" of the interaction
between the highly negatively charged region 3.2 with the highly
positively charged RNA exit channel (40, 41) would be expected to
reduce
70-core affinity which in fact is what has been
observed (24).