ABSTRACT
The kinetics of open complex formation were measured by migration retardation
assay and DNase I footprinting at the activator-dependent promoters
ara P1
,
lac P1
and
gal P1
. In each case, the rate of open complex formation was significantly faster if
the activator, AraC for
ara
and CAP for
lac
and
gal
, had been added before RNA polymerase. The results indicate that complexes of
transcriptional activators, RNA polymerase and promoter can exist in two
states, one which can form open complexes rapidly and one which cannot.
A derivative of the
araBAD
promoter,
p
BAD
I
2
-I
1
, can be significantly activated by AraC protein
in vivo
and
in vitro
(
1
). Here we show that the ability of the AraC-RNA polymerase-promoter complex to form a transcriptionally competent open
complex depends on the assembly order of the three components. This unexpected
property is not unique to the arabinose promoter. We find the same results with
CAP protein and RNA polymerase at the
lac
and
gal
promoters. While an order of assembly effect is theoretically possible for
systems containing as few as three components, it is a surprise to find it with
initiation complexes. Cells do not appear to possess a mechanism for
controlling assembly order. Therefore, our findings could be interpreted to
mean that
in vivo
two types of complexes form at some promoters, that additional and presently
unknown factors channel the assembly of initiation complexes so that only one
type of activator-polymerase-promoter complex forms or that inactive complexes are
disassembled. The potential problem raised by our findings appears to be more
important at promoters for which an activator binds slowly.
AraC protein was purified to homogeneity by Jeff Withey (
2
) and RNA polymerase holoenzyme as well as CRP was purified by Steve Hahn (
3
) and was >50% active (
1
).
The
ara P1
promoter is based on
P3-I
2
-I
1
(
1
,
4
). The
gal P1
promoter (
p16C
) was provided by Henri Buc (
5
). The
lac P1
promoter (
29C
) contains a point mutation that abolishes
lac P2
activity (
6
). The DNA fragments used for all experiments, which were ~250 bp long, with the polymerase binding site near the middle, were
amplified by PCR from plasmid DNA templates using
32
P-end-labeled primers. The PCR products were purified on 6% acrylamide
gels and electroeluted. When subjected to electrophoresis on denaturing gels,
the DNA formed single bands, with no indication that any appreciable fraction
was nicked. DNA stocks were kept at -20oC in TE buffer (10 mM Tris-HCl, 1 mM EDTA) containing 50 mM KCl.
The DNA migration retardation assay buffer contains 50 mM KCl, 25 mM Na-HEPES,
pH 7.4, 2.5 mM MgCl
2
, 2.5 mM dithioerythritol, 100 [mu]M cAMP, 100 [mu]g/ml bovine serum albumin, 0.1 mM K-EDTA, 5% glycerol, 1% arabinose and
0.05% NP-40. The proteins were incubated with DNA at 37oC. At each time point, 20 [mu]l of the reaction was withdrawn and mixed with 1 [mu]l heparin for 1 min and then loaded onto the gel. The final
concentration of heparin was 100 [mu]g/ml. Electrophoresis was at 5 V/cm for 2 h through 6% acrylamide, 0.1% bis-acrylamide horizontal submerged gels equilibrated with 10 mM Tris-acetate, pH 7.4, 1 mM K-EDTA. Buffer at 20oC was circulated through the apparatus, thereby
maintaining the gel at 20oC (
7
). Gels were dried and the radioactivity in bands quantitated with a
phosphorimager (Molecular Dynamics).
DNase I footprinting was done as previously described (
8
). Proteins were bound as described above, but in 50 [mu]l reaction volumes. At the times indicated, 1 [mu]l 50 mM CaCl
2
and 1 [mu]l 2 mg/ml DNase I was added for 20 s. Then 200 [mu]l quench solution (0.9 M NH
4
OAc, 50 mM Na-EDTA, 5 mg/ml calf thymus DNA and 0.2 mg/ml heparin) were added.
The quenched reaction was ethanol precipitated twice, lyophilized and
resuspended in 10 [mu]l of a 1:1 (v/v) mix of TE buffer and stop solution (95% formamide, 25 mM
EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol FF). The loading volume
was adjusted so that each sample contained approximately the same amount of
radioactivity. Samples were heat denatured and subjected to electrophoresis in
a 6% sequencing gel and gels were autoradiographed at -70oC with intensifier screens.
Addition of RNA polymerase to a solution containing AraC and DNA yields rapidly
forming open complexes as measured by the DNA migration retardation assay (Fig.
1
;
1
). When RNA polymerase is added before AraC, however, the kinetics of open
complex formation, following the initial burst on 10% of the molecules, are
much slower.
In the experiments described above, we found that the kinetics of open complex
formation on three different promoters is measurably faster if the activator
protein is bound to the DNA before the addition of RNA polymerase rather than
if RNA polymerase is added before the activator protein. Control experiments on
the
ara
operon show that polymerase that has been added before AraC binds at or near
the
ara
promoter and prevents the binding/activation of polymerase after the subsequent
addition of AraC. The DNA contains no obvious -10 and -35 sequences other than those at the
ara p
BAD
promoter. Secondary polymerase binding sites have been observed near some
promoters and their presence has complicated data interpretation (
9
-
11
). For the experiments done with
ara
two such sites, at positions -64 and -96, had been removed (
1
). We also used mutant
lac
and
gal
promoters, each thought to contain only single polymerase binding sites (
5
,
6
). Neither DNase I footprinting nor KMnO
4
footprinting of the
ara
DNA (
1
) revealed any polymerase binding sites or open complexes in the DNA other than
at the promoter.
In light of all the data, we conclude that RNA polymerase itself is capable of
binding to at least some of the promoters that normally require activator
proteins. This polymerase binds in an inactive state. Further, if activator
protein is subsequently added, the polymerase does not rapidly form an open
complex. Perhaps to form an open complex, the polymerase must dissociate and
then bind to an activator-DNA complex. Observation of effects as we have described here depend, of
course, on slow dissociation of the inactive polymerase molecules only after
the activator protein has been added. They say nothing about the dissociation
rate of polymerase from the promoter before the activators AraC or CAP have
been added. Effects analogous to what we have described here might be the
explanation for the frequently observed phenomenon that with some promoters
significantly less than 100% of the DNA template molecules can be utilized in
single round transcription experiments.
We thank Sydney Kustu, John Little, Wilma Ross, Steve Busby and Andrew Travers
for comments on an earlier manuscript describing most of this material, Jeff
Withey for technical assistance, Thadd Reeder for initiating the project and
members of the laboratory for ongoing discussions and comments on the
manuscript. This work was supported by National Institutes of Health grant
GM18277 to RFS.
REFERENCES
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