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J. Biol. Chem., Vol. 278, Issue 32, 29701-29709, August 8, 2003
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From the Division of Biological Sciences and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0634
Received for publication, May 2, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-family
initiation subunit gp55. The T4 late promoter only opens above 15–20
°C, but once it has been formed remains open and transcriptionally active
for days at –0.5 °C. The low temperature-trapped open complex and
its isothermally formed state are shown to be structurally distinctive. Two
"extended –10" 70 promoters, which, like
the T4 late promoter, lack "–35" sites, have been subjected
to a comparative analysis: the T4 middle promoter PrIIB2 opens and
closes thermoreversibly under conditions of basal and MotA- and AsiA-activated
transcription. The open galP1 promoter complex, whose transcription
bubble is very AT-rich, also closes reversibly upon shift to –0.5
°C, but more slowly than does the rIIB2 promoter.
Formation of a trapped-open low temperature state of the promoter complex
appears to be a singular property of gp55-RNA polymerase holoenzyme. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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15
bp of DNA extending from bp –11 beyond the transcriptional start site;
the transcribed and non-transcribed DNA strands become widely separated upon
promoter opening
(1–3).
In general, promoter complexes open with increasing temperature and close when
the temperature is lowered; in hyperthermophilic bacteria, for example,
promoter opening requires elevated temperatures. 4) In the presence of
nucleotides (nt),1
formation of initial transcripts comprising the first few internucleotide
linkages generally follows rapidly. Initial steps of RNA synthesis are not
processive; short aborted transcripts are produced at all promoters and can be
overwhelmingly predominant at some. 5) RNA polymerase clears the promoter by
means of processive RNA chain elongation.
The production of transcripts at different promoters can be controlled at any of these steps: Activity can be limited at the first step if RNA polymerase affinity for the promoter as duplex DNA is low. The equilibrium at step 2 can be unfavorable, and this step can also be kinetically limiting. Step 3 is generally rapid, but the equilibrium can be unfavorable. The transition to the final step of processive transcript elongation and promoter clearance is limiting at certain promoters at which initial transcripts are repetitively but abortively produced. Individual equilibrium and rate constants associated with these steps are, of course, dependent on environmental variables. Depending on temperature, different steps can present the principal barrier to transcriptional activity at a single promoter (4–22). Activators of bacterial transcription can exert their effects by intervening at any one, or at more than one, step. Some activators (such as CRP, the cAMP receptor protein) activate transcription at different promoters by affecting different steps (e.g. Refs. 10 and 23).
The reaction sequence that generates the open, initiation-ready promoter
complex is adequately described by three linked equilibria shown in Reaction
1,
 | (Eq. 1) |
is the RNA polymerase holoenzyme; P is the promoter;
C1 is the initially forming closed promoter complex; C2
is an isomerized but still closed intermediate; Co is the
strand-separated open complex, with associated equilibrium constants
K1, K2, and K3,
and forward and reverse rate constants k1,
k–1 etc. (see Refs.
9 and
18 for relatively recent
reviews). The following complications are encountered in inferring structures
of the C1 and C2 intermediate complexes:
70-RNA polymerase holoenzyme complexes with the phage
PR and lacUV5 promoters do not accumulate the
C2 complex as the predominant equilibrium or isothermal steady
state component (5,
20,
24). Access to analysis of
intermediates is also provided by rapid-kinetic pre-steady state methods
(25) or by temperature
downshift to transiently accumulate an intermediate component ahead of a
(kinetically) slow step
(4–6,
8).
A distinctive example of irreversibility in promoter interactions has also
been noted recently in an analysis of the mechanism of activation of the late
genes of bacteriophage T4
(26). The promoters of these
genes consist of a simple TATA box (consensus: TATAAATA) serving as an
extended –10 site; there is no –35 site or required specific
sequence upstream of the T4 late TATA box
(27,
28). Recognition of T4 late
promoters is conferred on the host RNA polymerase core by gp55, the initiation
subunit encoded by T4 gene 55. The small (185 amino acids) gp55 has been
proposed as a distantly related member of the 70 family of
transcription factors that lacks the protein domain that would interact with a
–35 site (29,
30). The gp55 segment thought
to be related to 70 homology segment 2 does interact with
E. coli RNA polymerase core
(31) and mutagenesis
identifies a gp55 segment related to 70 homology segment 2.2
as an RNA polymerase core binding site
(32).
Activation of T4 late transcription by the T4-encoded sliding clamp
replication-processivity factor requires the co-activator gp33 and depends on
direct interactions of gp33 and gp55 with the sliding clamp (Refs.
33 and
34; reviewed in Ref.
35). T4 late promoter opening
by the gp55-RNA polymerase holoenzyme is thermoirreversible: the late promoter
does not open at 0 °C, but once opened at 30 °C remains open after
shift to the lower temperature
(26). At a hybrid promoter for
gp55- and 70 holoenzymes, only gp55 confers
thermoirreversibility of promoter opening.
The experiments that are described below pursue the further analysis of
this apparently locked-in open promoter. The low temperature-open promoter
complex is shown to be transcriptionally competent and extremely long-lived
over a range of temperatures. Probing its stability with the competitively
binding polyanion heparin and probing its transcription bubble with
KMnO4 indicates that the trapped, low temperature-open complex and
the open complex formed isothermally at 30 °C are structurally
distinctive. These properties of the gp55 holoenzyme promoter complex have
been placed in the context of the more studied 70 holoenzyme
by a parallel analysis of open complexes at two "extended
–10" 70 promoters. (These are promoters that
also lack a significant –35 promoter element.) The properties of these
two 70 holoenzyme open promoter complexes are shown to
differ from those of the open T4 late promoter complex. 1) The T4 middle
promoter PrIIB2 opens and closes thermoreversibly. 2) The
galP1-35 promoter
(36) also closes at 0 °C
once it has been opened at 30 °C, but this extremely AT-rich and readily
melting promoter (36,
37,
38,
39) does so more slowly. The
analysis suggests that the gp55-RNA polymerase holoenzyme differs from
70-RNA polymerase holoenzyme in allowing the generation of a
trapped-open state of its cognate promoters.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-(1–565) was
a generous gift from K. Severinov. The 568 bp PCR fragment used for
KMnO4 footprinting of bacteriophage T4 late promoter
(P23) complexes was generated from placO-SK110-rrnB(T1+T2)
(26). A 653-bp PCR fragment
from pRT510-C+18 (41), which
contains a hybrid gp55- or 70-dependent promoter, was used
for analysis of transcription. In the presence of ATP, GTP, UTP, and UpG
dinucleotide, this construct allows formation of an initial 18-nt transcript;
a 427-nt transcript is made in the presence of all four NTPs. The 214-bp PCR
fragment used for footprinting and transcription analysis of the bacteriophage
T4 middle promoter (PrIIB2) was generated from plasmid pSTS416
(42). This template allows
formation of a 120-nt run-off transcript. The 127-bp DNA fragment used for
footprinting and transcription analysis of the galP1 promoter and the
115-bp fragment from galP1-35 were generated from plasmids pAA121 and
its derivative with other sequence substituted for the UP-element,
respectively; these two plasmids were kindly provided by S. Minchin and S.
Busby (38,
39). DNA sequences of these
promoters are presented in Fig.
1.
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Formation and Analysis of Promoter Open Complexes—Standard
procedures for assembly of promoter complexes have been described
(26). Promoter DNA (30 and 100
fmol for footprinting and transcription, respectively) and proteins (1 pmol of
RNA polymerase core, 6 pmol of gp55 or 70) were mixed in a
15-µl reaction buffer containing 200 mM potassium acetate, 33
mM Tris acetate (pH 7.8), 10 mM magnesium acetate, 150
µg/ml bovine serum albumin, 1 mM dithiothreitol, 0.05% (w/v)
Brij58, and 5% (w/v) polyethylene glycol (PEG)3300. RNA polymerase core enzyme
from uninfected or T4-infected E. coli was used as specified for the
individual experiments described below. Promoter complexes were formed for 60
min at 30 °C, then shifted to low temperature for 30 min or for the time
noted (Fig. 2) before further
analysis. Maintaining temperatures not exceeding 0 °C was assured by
performing low temperature manipulations in a 3.5 °C cold room, using a
constant temperature bath set to –0.5 °C.
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KMnO4 Footprinting—Reaction
mixtures containing promoter complexes in a 15-µl volume were mixed with 2
µl of KMnO4 (to 10 mM final concentration for
analysis of T4 late promoter complexes and 6 mM for T4 middle and
galP1 promoter complexes). Oxidation was stopped after 30 s by adding
nine volumes of stop solution containing 200 mM
-mercaptoethanol. Samples were extracted with phenol/chloroform/isoamyl
alcohol, DNA was precipitated with ethanol, dried, reacted with 10% (v/v)
piperidine/1 mM EDTA at 90 °C, re-extracted with phenol, and
re-precipitated, as described
(26). Equal quantities of
radioactivity were loaded for resolution by denaturing PAGE, visualized by
phosphorimaging and quantified, as described
(26).
Transcription—All transcription experiments assayed single
rounds of RNA synthesis. For formation of 19-mer transcripts initiating at the
hybrid T4 late promoter (Figs.
1,
4C, and
6), open complexes in 15 µl
were mixed with 5 µl of NTPs in reaction buffer, yielding final
concentrations of 100 µM UpG, 5 µM each of ATP,
GTP, and [
-32P]UTP, and 100 µM 3'
O-methyl CTP (250 µM for the experiment shown in
Fig. 4C). Reactions
carried out at 30 °C contained 100 µg/ml heparin; heparin was omitted
for transcription at –0.5 °C. RNA synthesis was terminated 10 min
after addition of nucleotides. For long time course experiments
(Fig. 6), samples were
incubated for specified times at –0.5 °C, then transferred to 30
°C for 1 min prior to addition of NTPs and heparin for 10 min. Samples
were processed, resolved on 23% polyacrylamide gel (37.5:1 acrylamide/bis),
analyzed, and quantified by phosphor-imaging. For RNA chain elongation
(Fig. 5), the 18-mer initial
transcript was formed in the presence of 100 µM UpG, 5
µM each of ATP, GTP, and [-32P]UTP for 10 min
at 30 or –0.5 °C, all four unlabeled NTPs were added to final
concentrations of 800 µM, and the reaction was sampled at
various times thereafter. RNA was prepared and analyzed as specified above,
except that 12% polyacrylamide gel was used to resolve transcripts. For the
experiment shown in Fig.
6C, the reaction buffer did not contain polyethylene
glycol.
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Run-off transcription initiating at PrIIB2 was carried out with
1 mM ATP, 1 mM GTP, 0.1 mM UTP, 0.1
mM [-32P]CTP, and 100 µg/ml heparin; heparin
was omitted for transcription at –0.5 °C; run-off transcripts were
resolved on 6% polyacrylamide gel. Run-off transcription initiating at the
galP1 promoters was carried out with 1 mM ATP, 1
mM UTP, 0.1 mM GTP, 0.1 mM
[-32P]CTP, and 100 µg/ml heparin, but heparin was omitted
for transcription at –0.5 °C. Run-off transcripts were resolved on
10% polyacrylamide gel. For long time course experiments with the
galP1-35 promoter (Fig.
8C), samples were incubated for appropriate times at
–0.5 °C, then transferred to 30 °C for 0.5 min prior to addition
of NTPs and heparin for 6 min.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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70-RNA polymerase
holoenzyme at the lacUV5 and PR promoters melt a
15-bp DNA segment thermoreversibly
(5,
9,
18,
20). This feature has been
utilized to trap and analyze intermediate states of closed promoter complexes
(6,
8,
21). In contrast, it was
recently found that the bacteriophage T4 late promoter open complex formed by
gp55-RNA polymerase holoenzyme remains open upon shift to lower temperature
(26). Further analysis of the
P23 late promoter open complex by KMnO4 footprinting
reveals a stable open conformation that forms rapidly upon temperature shift
from 30 to 0 °C (Fig. 2).
The low temperature state of the T4 late promoter open complex is
characterized by overall lower KMnO4 reactivity in the promoter
bubble, together with nearly complete loss of reactivity of T–12 in the
transcribed (template) strand (Fig.
2). The T4 late promoter open complex withstands challenge with
heparin at 25 °C or 30 °C, but the low temperature-open complex
dissipates rapidly in the presence of 100 µg/ml heparin
(Fig. 2). The T4 late promoter opens slowly in the absence of its co-activator and sliding clamp activator at 30 °C, under the conditions of these experiments (26). This makes it possible to use a temperature downshift/upshift sequence to determine whether the quantitative changes of KMnO4 reactivity at –0.5 °C seen in Fig. 2 reflect a partial dissociation of promoter complexes, and whether these low temperature complexes are trapped in an inactive form (Fig. 3). Open promoter complexes were allowed to form at 30 °C for 60 min (lane 1), shifted to –0.5 °C for 30 min (lane 2), then shifted back to 30 °C for a time interval that is too brief to allow appreciable de novo formation of open complexes at this concentration of RNA polymerase holoenzyme (1 and 2 min, lanes 3 and 4, respectively) (26) and assayed at each step by treatment with KMnO4. Reactivity to permanganate was very rapidly and fully restored upon shift back from –0.5 to 30 °C, indicating that the low temperature and "conventional" (30 °C) states of the T4 late open promoter complex are in rapidly established equilibrium. In contrast, the low temperature-open complex is not formed upon continuous incubation at –0.5 °C on the time scale of the experiment (up to 1.5 h; Fig. 2A, lane 9), or even in 48 h (as we show below).
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The low temperature open complex is also transcriptionally competent.
Promoter escape, detected by disappearance of the promoter transcription
bubble, followed upon addition of NTP substrates
(Fig. 4, A and
B). The open complex held at –0.5 °C for 30 min
also fully retained the ability to make the appropriate 19-mer transcript at
the low temperature when presented with UpG, ATP, GTP, UTP, and
chain-terminating 3' O-methyl CTP
(Fig. 4C, compare
lanes 8, 9, and 10 with lane 1), although it lost
this capacity rapidly when challenged with heparin before nucleotide addition
(compare lanes 3–7 with lanes 8–10), due to
disruption of this low temperature open complex
(Fig. 2). Indeed, the 18-mer
initial transcript elongated at –0.5 °C upon addition of CTP to the
three other rNTPs, although elongation was quite slow (0.1–0.4
nt/s) at this low temperature (Fig.
5). For this experiment, open promoter complexes were formed at 30
°C for 60 min, and shifted to –0.5 °C for 30 min before adding
UpG, ATP, GTP, and [-32P]UTP for 10 min to make the 18-mer
initial transcript (Fig. 5,
lane 6). All four unlabeled NTPs were added (with 800
µM UTP to dilute out the label), and samples were taken at the
times noted in order to analyze the resulting first cycle of RNA chain
elongation (lanes 7–11). As a control, the elongation process
was also followed at 30 °C on an appropriately shorter time scale
(lanes 1–5). Clearance of the promoter from its +18 start was
at least as effective at –0.5 °Casat30 °C (compare lanes
6 and 11 with lanes 1 and 2, respectively).
There were no prominent sites of transient pausing during elongation at
–0.5 °C (compare lane 11 with lane 2, for
example).
Transcription assays were used to examine the barrier to reversible
promoter closing on a considerably extended time scale and at different
temperatures. Persistence of the open promoter complex after shift to low
temperature was measured by ability to form a 19-mer initial transcript in the
presence of heparin (Fig.
4C) after a very brief shift back to 30 °C allowing
thermal equilibration (Fig. 3)
but almost no de novo formation of open complexes under these basal
conditions (Ref. 26, and
confirming data, not shown). The loss of polymerase activity during these
excessively long incubations at low protein concentration was assessed in
parallel by shifting an aliquot of the reaction mixture back to 30 °C for
1 h to allow formation of open complexes by all RNA polymerase molecules
retaining activity. Separate experiments verified that the late promoter does
not open (under basal conditions, in this reaction medium) in 48 h at
–0.5 °C, in 43 h at 6 °Corin23hat9 °C
(Fig. 6A and data not
shown). At intermediate temperatures of 12 and 15 °C, residual opening was
observed after 7 h of incubation; the promoter opened slowly and progressively
at 20 °C (30% opening after 7 h) (data not shown). The open promoter
did not close in this long term time frame after shift from 30 °C to
–0.5 °C (23 h, Fig.
6B, and even 43 h, data not shown), or in 23 h after
downshift from 30 °C to 6, 9, or 12 °C (data not shown). Omission of
polyethylene glycol (PEG 3300) from the reaction medium did not make promoter
opening reversible: most open complexes remained open 23 h after shift from 30
to –0.5 °C (Fig.
6C).
Most promoters that are recognized by 70 family
initiation subunits have the characteristic –35 and –10 DNA sites.
T4 late promoters are exceptional in lacking a –35 site, and gp55 is
correspondingly exceptional in lacking any counterpart of the
70 family protein domain that interacts with the –35
site (structure domain 4; sequence homology segment 4.2)
(43,
29,
30,
44,
45). A hybrid promoter for
gp55- and 70 holoenzyme has been constructed by placing the
–35 70 consensus site upstream of the T4 late promoter
(41)
(Fig. 1). The gp55 holoenzyme
keeps this gp55/70 hybrid promoter open after temperature
downshift from 30 °C to 0 °C, while the 70
holoenzyme opens and closes the same promoter thermoreversibly between 30 and
0 °C (26). It was
previously suggested that the distinction between the properties of these two
holoenzymes might be due to differences of interaction with the upstream
promoter site. The next experiments explore this conjecture.
T4 middle promoters are members of the "extended –10"
subclass of 70 promoters (consensus: TGXTATAAT; the
TG extension of the –10 site is recognized by 70
homology segment 2.5). Middle promoters lack the 70
–35 recognition site but substitute a binding site centered around bp
–30 for their activator, MotA. AsiA, the co-activator of T4 middle
transcription, binds to 70 at two sites located in homology
segments 4.1 and 4.2 (structure domain 4) and also to MotA
(thereby dissociating dimeric MotA into the monomer). MotA additionally
interacts with the very C terminus of 70
(46–50).
The thermoreversibility of opening the T4 middle promoter PrIIB2
with 70-RNA polymerase holoenzyme was examined under
conditions of basal and MotA/AsiA-activated transcription. When the
rIIB2 open promoter complex was shifted from 30 to
–0.5 °C for 30 min, KMnO4 footprinting revealed less than
10% of residual reactivity (Fig.
7A). AsiA, alone or together with MotA, did not affect
the thermoreversibility of promoter opening
(Fig. 7A). A
transcription experiment showed that the previously opened
rIIB2 promoter closes within 30 min at 0 °C
(Fig. 7, C and
D). E. coli RNA polymerase core is modified
after phage T4 infection by ADP-ribosylation of its -subunits in the
C-terminal domain that interacts sequenceselectively with AT-rich DNA upstream
of the core promoter. Binding to this UP element is eliminated by
ADP-ribosylation of (at Arg-265). ADP-ribosylated (T4-modified) RNA
polymerase was also seen to open and close the rIIB2
promoter thermoreversibly (Fig. 7,
C and D).
The effect of deleting region 4.2 of 70 on
thermoreversibility was also examined. Basal (MotA- and AsiA-independent)
transcription at PrIIB2 was dramatically decreased when
70 was replaced with 70-(1–565). It
has been shown that AsiA becomes a strong activator of T4 middle transcription
in the context of the segment 4.2-deleted 70-(1–565)
(51). Promoter opening at 30
°C was correspondingly rescued by AsiA and not further affected by MotA
(Fig. 7B).
Thermoreversibility was not qualitatively affected by the presence of AsiA or
MotA (although it is conceivable that MotA has a small quantitative effect on
residual promoter opening after shift to –0.5 °C)
(Fig. 7, A and
B).
In summary, promoter opening at the "extended –10" T4
middle promoter PrIIB2, which has no –35 promoter element, by
E. coli 70-RNA polymerase holoenzyme is
thermoreversible. Eliminating residual nonspecific DNA interaction at the
upstream side of the promoter complex by removing the 70
homology segment 4.2 or by ADP-ribosylation of the C-terminal DNA-binding
domain of the RNA polymerase -subunit does not make promoter opening
irreversible. MotA and AsiA, the activator and co-activator of T4 middle
transcription, do not qualitatively change the reversibility of promoter
opening.
The analysis of extended –10 promoters also included the extensively
analyzed galP1 promoter, which has a relatively strong UP element
(37,
36,
38,
52,
39), and its derivative
galP1-35, which removes that element by substituting other sequence
upstream of bp –35 (38).
The galP1 promoter opens at a relatively low temperature
(39), and replacing the UP
element further lowers the characteristic promoter opening temperature
(38). Under the conditions of
our experiments, the galP1 and galP1-35 promoters did not
open in 90 min at –0.5 °C (Fig.
8A), but clearly remained open after 30 min at that low
temperature once they had been opened for 60 min at 30 °C. The
distribution of permanganate reactivities at T+2, –2, –3,
–4, and –6 was seen to be approximately the same at 30 °C and
after shift to –0.5 °C, thus giving no indication of partial
promoter closure at the lower temperature
(Fig. 8A), as is the
case for the T4 late promoter, for which permanganate reactivity at T–12
was lost upon temperature downshift (Fig.
2). Indeed, the galP1 and galP1-35 open
complexes retain a high proportion of activity in a single cycle of
transcription after 30 min at –0.5 °C
(Fig. 8, B and
D). A difference in the lengths of the 45-nt run-off
transcripts made at the two temperatures can be noted in
Fig. 8B, and probably
corresponds to a progressive shift previously noted for transcription at
temperatures below 37 °C (Fig. 5 of Ref.
39). Substituting the
ADP-ribosylated T4-modified RNA polymerase core, which should generally
decrease specific (UP element) as well as nonspecific DNA interactions of the
two -subunit C-terminal domains, had no effect on the retention of
transcription activity after shift to –0.5 °C for 30 min
(Fig. 8, B and
D).
The retention of strong transcription activity of the low temperature-open promoter complex is (somewhat) at variance with a prior observation that the galP1 promoter is transcriptionally inactive at low temperatures at which partial but substantial promoter opening is detected by KMnO4 footprinting (39). A mobile equilibrium between transcriptionally competent open and inactive closed states of a promoter should be driven toward complete opening upon addition of nucleotide substrates, consistent with the results shown in Fig. 8, B and D. The contrary prior observations came from an experiment in which transcription initiated in the presence of heparin and in the presence of a relatively low concentration of UTP: UMP is the second nucleotide of the galP1 transcript and its effective Km may be temperature-dependent; the stability of open galP1 promoter complexes at low temperature to challenge by heparin was also not reported as having been directly examined in the prior work (but compare with Fig. 2). Because the relative transcript yield at –0.5 °C was seen to diminish at low NTP concentrations (data not shown), high nucleotide concentrations were used for the experiment presented in Fig. 8B (in particular, 1 mM each of ATP and UTP, which are incorporated at the first 2 steps of RNA chain elongation).
The apparent trapping of the 70 holoenzyme complex with
the galP1-35 promoter in a low temperature-open state was analyzed
further at longer time scale and at different temperatures. Under the
conditions of these experiments (see "Materials and Methods"),
galP1 opened sufficiently slowly at 30 °C to allow use of the
experimental design shown in Fig.
6: promoter opening at –0.5, 6, and 10 °C was assayed
after shift to 30 °C for 0.5 min (allowing temperature equilibration but
barely any formation of new open complexes; data not shown); retention of
polymerase activity during long incubations at these lower temperatures was
assayed by shifting back to 30 °C for 60 min
(Fig. 8, C and
E). The galP1-35 promoter failed to open in 23 h
at –0.5 °C, but slowly and only partially opened at 6 °C (in 23
h), and eventually opened almost completely at 10 °C (in 23 h). In
contrast to the pre-opened T4 late promoter, which remained open after
temperature downshift and long term incubation
(Fig. 6B), the
pre-opened galP1-35 promoter closed, albeit slowly, after long term
incubation at –0.5 °C (Fig. 8,
C and E). At 6 °C, the pre-opened promoter
closed slowly and only partially, consistent with eventually reaching a
partially open state at equilibrium. Pre-opened complexes remained open after
shift down to 10 °C, consistent with slow establishment of an open state
at equilibrium, in this reaction medium.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thus, it is possible that the open promoter complex undergoes some
structural change upon shift from 30 °C to –0.5 °C. However, if
distinct states of the open complex exist at the higher and lower temperature,
they are rapidly interconverted upon temperature downshift
(Fig. 2A) and upshift
(Fig. 3A) under
conditions in which the de novo formation of open complexes is quite
slow. The low temperature-open complex is transcriptionally fully active at
–0.5 °C (Fig.
4C); elongation of RNA chains through a 400-bp
transcription unit is slow but relatively steady without prominent sites of
pausing (Fig. 5).
How does this irreversibility of promoter opening arise, and where does the
block to establishing equilibrium reside? In terms of the standard kinetic
model stated in the introduction (Reaction 1): a) Is it in the forward
direction of promoter opening, either in converting C1 to the
C2 closed complex or in the C2 
Co
promoter opening step? A kinetic block solely in the forward direction implies
that the equilibrium state of the basal promoter complex at –0.5 °C
is open. However, the late promoter does not open in 48 h at –0.5 °C
or even in 23 h at 6 or 9 °C, and only barely opens in 7 h at 15 °C.
It is overwhelmingly likely that the equilibrium state of the basal promoter
complex at –0.5 °C is closed. b) Does the kinetic barrier to
establishment of equilibrium operate on the reverse direction? A block here
might be generated by a very sharp temperature-dependence of conversion of the
open complex to the C2 closed complex. However, the late promoter
also does not close reversibly at 6, 9, or 12 °C. That makes it
exceedingly unlikely that this is the simple explanation of irreversible
retention of promoter opening at –0.5 °C.
With evidence lacking for either of the preceding explanations, one is left with less simple alternatives. It appears that the open promoter complex may exist in multiple forms: we propose that the C2 closed complex opens reversibly into an open complex Co(1), which converts into a trapped-open form Co(2). Our limited explorations have failed to uncover an "escape" pathway from the trapped-open Co(2) state by varying temperature. Removing the macromolecular crowding agent, PEG, which might stabilize otherwise mobile structural states of the promoter complex (54), from the reaction medium also does not eliminate trapping.
The T4 late promoter consists solely of an 8 bp-long "extended
–10" element. Whether the lack of a –35 element is
sufficient to generate thermoirreversible promoter opening has been explored
by examining two "extended –10" 70
promoters that also lack –35 elements: the T4 middle promoter
PrIIB2 and the galP1 promoter. Opening of the T4
rIIB2 middle promoter is fully reversible between 30 and
–0.5 °C, regardless of whether the promoter functions in its basal
mode or in conjunction with its MotA activator and AsiA co-activator
(Fig. 7A). One might
reason that 70 homology segment 4.2, which recognizes the
–35 (promoter) site, must also have some nonspecific affinity for
upstream DNA and could mediate reversible promoter closing, but removing
segment 4.2 does not prevent promoter closing when the temperature is shifted
down to –0.5 °C (Fig.
7B). Similarly, blocking DNA binding of the -CTD
by ADP-ribosylation, a T4 phage infection-associated modification, does not
eliminate reversibility of opening at the rIIB2 promoter
(Fig. 7, C and
D).
The galP1 promoter was selected for comparative analysis because
of its ability to open at low temperature in linear DNA
(37,
36,
38,
39): partial promoter opening
has been noted at temperatures as low as 6 °C, in some experiments
(39). The transcription bubble
of the galP1 promoter open complex is especially AT-rich, with 9 out
of 10 A:T base pairs between –7 and +3. Opening galP1 at the
low temperature does not require the T/A-rich UP element-like cluster that is
replaced in galP1-35 (Fig.
1). Conforming the –10 site to its consensus (TATGGT
TATAAT) or introducing a consensus –35 site (TTGACA) further lowers the
promoter opening temperature
(36,
38), but removing the TG
extension of the –10 site (TG TT) and introducing a –35
site (to create the promoter designated galPcon) markedly
increases the temperature at which the promoter opens
(39). Two additional
observations with this collection of promoter constructs motivate a direct
comparison with the T4 late promoter. (a) The galP1 promoter
was reported to be transcriptionally inactive or seriously reduced in activity
at low temperatures (6 and 14 °C, respectively) at which it was
respectively partly or almost completely open. Promoter opening at very low
temperature without concomitant transcriptional activity has also been
reported to result from deleting a large internal segment of the RNA
polymerase -subunit
(55). (b) An
observation indicating apparently irreversible promoter opening was reported
for the galPcon promoter (a conventional
70 promoter with –35 and –10 sites and no TGx
extension of the –10 site): after it has been opened at 37 °C, this
promoter retains transcriptional activity (for 30 min) after shift to a lower
temperature at which it does not open (in 30 min; assayed with
KMnO4) (39).
In the reaction medium that allows a direct comparison with the T4 late promoter, galP1 and galP1-35 do not open at –0.5 °C, but remain open (for 30 min) after having first been opened at 30 °C (Fig. 8A), and initially retain transcriptional activity at the low temperature (Fig. 8, B and D). However, qualitative as well as quantitative differences between the low temperature-open complexes at the T4 late and galP1 promoters are seen when the analysis is extended to longer time scales (Fig. 8, C and E). Given sufficient time, the galP1-35 promoter closes (Fig. 8E), just as the T4 rIIB2 promoter does. GalP1-35 opens completely but slowly at 10 °C, so it is not surprising to find the promoter remaining open at 10 °C after downshift from 30 °C; at 6 °C galP1-35 opens and closes slowly (and is probably only partly open at equilibrium). These properties of the galP1 promoter are adequately explained in simple terms (Reaction 1, see the Introduction): temperature dependence of rates of opening and closing, and temperature dependence of the equilibrium state of this low temperature-melting promoter. Thus, in our view, the rIIB2- and galP1-extended –10 promoters do not differ mechanistically in regard to promoter opening and closing, although the T4 rIIB2 promoter clearly closes much more rapidly than the gal promoter does at –0.5 °C (possibly as a result of sequence and nucleotide composition differences between these two promoters in the DNA segment that opens into the transcription bubble).
It is only the open complex of the gp55 holoenzyme with the T4 late
promoter that has been found to be capable of converting to a trapped-open
state, but we suspect that this cannot be a property solely of gp55. The
transcription bubble traverses the core - and '-subunits,
and strand separation at the downstream end of the transcription bubble is
maintained by interaction with - and '-subunits
(2,
3). Three 70
promoters, one with a –35 site
(26) and two without one (this
work) open and close thermoreversibly. It is conceivable that
70 constrains polymerase core or DNA indirectly to prevent
the structure change that traps a low temperature-open state. Screening of
sigma and core mutants (in conjunction with a promoter that does not open at
low temperature) may well yield further insights in this regard. It would also
not surprise us if open promoter complexes with duplex DNA, even those that
close reversibly at 0 °C, could be generally trapped by rapid quenching to
sufficiently low temperatures. This could prove useful for structure
studies.
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FOOTNOTES |
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To whom correspondence may be addressed. Tel.: 858-534-2451; E-mail:
mkamali{at}biomail.ucsd.edu.
To whom correspondence may be addressed. Tel.: 858-534-3029; Fax:
858-534-7073; E-mail:
epg{at}biomail.ucsd.edu.
1 The abbreviation used is: nt, nucleotides.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
KMnO4 reactivity at –0.5 °C in the absence of heparin;
(•) KMnO4 reactivity at –0.5 °C in the presence of
100 µg/ml heparin.
