(Received for publication, April 17, 1995)
From the
The loop at the 52-position of the cAMP receptor protein (CRP)
has been suggested as a potential site for contacting RNA polymerase on
Class II promoters where the CRP binding site is located at position
-41.5 (Bell, A., Gaston, K., Williams, R., Chapman, K., Kolb, A.,
Buc, H., Minchin, S., Williams, J., and Busby, S.(1990) Nucleic
Acids Res. 18, 7243-7250). Using protein-protein
photo-cross-linking, evidence is presented showing that the 52-loop of
CRP is in physical proximity to the subunit of RNA polymerase
holoenzyme. This interaction required the presence of a functional
preinitiation complex. The CRP suppressor mutation, K52N, increased the
efficiency of cross-linking, indicating an improved physical
interaction between the CRP 52-loop and the
subunit. Evidence
for direct interaction between the CRP 156-162 loop and
subunit of RNA polymerase on both gal and lac promoters are also provided. The data indicate that CRP bound to
the gal promoter contacts both the
and
subunits of RNA polymerase.
CRP ()is a dimeric protein which binds to consensus
sites on the promoter through a helix-turn-helix motif in its
C-terminal domain. The CRP target sites on DNA are located at a variety
of positions with respect to the transcription initiation site.
Typically, Class I promoters (like lac P1) have the CRP
binding site at position -61.5, while that of Class II promoters
(like gal P1) is located at position -41.5.
Evidence
indicates that contact between CRP and RNA polymerase is involved in
transcription activation at the lac promoter. DNase I
footprinting demonstrated that CRP and RNA polymerase cooperatively
bind to the lac promoter (Li and Krakow, 1985; Ren et
al., 1988; Straney et al., 1989). Fluorescence
polarization experiments showed that CRP and RNA polymerase interact in
solution (Heyduk et al., 1993). CRP ``positive
control'' mutants which affect transcription activation without
altering specific DNA binding and bending have been isolated; they map
on an exposed loop spanning amino acid residues 156 to 162 in the
C-terminal domain (Bell et al., 1990; Eschenlauer et
al., 1991; Zhou et al., 1993). C-terminal truncated
mutants of the subunit of RNA polymerase cannot be activated by
CRP on the lac promoter (Igarashi et al., 1991; Kolb et al., 1993). Sharif et al. (1994) showed that
monoclonal antibodies directed against the
subunit inhibited
transcription from the CRP-dependent lac P1 promoter but not
from the lac UV5 and gal promoters. CRP activates
transcription from Class I promoters by contact of a primary activation
site in its C-terminal domain (amino acid residues 156-162) with
the C terminus of the
subunit of RNA polymerase. Protein-protein
photo-cross-linking has provided direct evidence that the CRP 161
position contacts the C-terminal domain of the
subunit of RNA
polymerase (Chen et al., 1994).
The mechanism of
transcription activation on Class II promoters is less clear. Evidence
suggests that the contact site for CRP on Class II promoters differs
from those on Class I promoters. Mutations in the subunit of RNA
polymerase abolish CRP activation for Class I promoters with no effect
on Class II promoters (Igarashi et al., 1991; Igarashi and
Ishihama, 1991; Zou et al., 1992). C-terminal truncations in
the
subunit impair the ability of RNA polymerase to respond to
CRP activation for gal transcription, suggesting that the RNA
polymerase activation site for CRP on a Class II promoter might be
located within the deleted C-terminal domain of
(Kumar et
al., 1994). Point mutations at position 52 (K52N,K52Q) restore the
CRP stimulatory activity of 156-162 pc mutants (H159L,
G162C,
159) on the gal promoter but not on lac (Bell et al., 1990; Williams et al., 1991).
These observations suggest that there are at least two different sites
on CRP which contact RNA polymerase at two different positions
depending upon the promoter class involved. Numerous studies of the lac promoter show that the surface-exposed CRP loop consisting
of amino acids 156-162 (Activating Region I) makes direct contact
with a site on the C terminus of the RNA polymerase
subunit. In a
recent study, Busby and his collaborators (Attey et al., 1994)
found that
protects upstream sequences both in the binary RNA
polymerase
gal P1 and ternary RNA
polymerase
CRP
gal P1 complexes. In the latter
complexes,
appears to make direct contact with the Activating
Region I, spanning amino acids 156-162, of CRP.
In this
report, we provide evidence that CRP bound to the gal promoter
contacts both the and
subunits of RNA
polymerase.
CRP stimulates transcription on a Class I promoter by contact
of its activating region at the 156-162 loop with the C-terminal
region of the subunit of RNA polymerase. Another surface-exposed
loop at position 52 on CRP is also considered a potential candidate for
contacting RNA polymerase during Class II promoter activation. To
directly study possible contact with RNA polymerase, site specific
photo-cross-linking was carried out using benzophenone-4-maleimide as a
sulfhydryl-specific photoreactive cross-linker.
The reactive
cysteine 178 of CRP was first replaced with serine (Pendergrast et
al., 1992), and a new cysteine was introduced into the CRP 52-loop
at position 56. Another mutant was constructed having the K52N
substitution in addition to its G56C substitution. The two CRP mutants
constructed for photo-cross-linking were C178S,G56C and
C178S,K52N,G56C. Transcription activation was determined in vivo by assaying -galactosidase activity. Both mutant CRPs retain
the ability to activate transcription on both lac and gal type promoters (Fig. 1). The mutant CRPs were purified and
reacted with benzophenone-4-maleimide (BPM) to specifically modify the
solvent-accessible cysteine residue on CRP introduced by site-directed
mutagenesis into the 52-loop at the 56-position.
Figure 1:
A, in
vivo expression of the lac operon. -Galactosidase assays
show the stimulating activity of mutant and wild-type CRPs which were
expressed in E. coli XE64.2 cells. B, in vivo expression of the gal-like class II promoter CCpmelR.
-Galactosidase assays show the stimulating
activity of mutant and wild-type CRPs which were expressed in E.
coli M182 cells (
crp39,
lac) with the
co-transformation of CCpmelR promoter in
pRW2.
To assess the
ability of the BPM-CRP to contact RNA polymerase, photo-cross-linking
was carried out on the (BPM-CRP)DNA
RNA polymerase ternary
complex. After UV irradiation, DNase I was added to digest the promoter
DNA, and the mixture was resolved on a SDS-10% polyacrylamide gel.
Western blotting with monoclonal antibodies specific for CRP,
,
,
`, and
was carried out to detect possible
cross-linked products. The results indicated that BPM-CRP cross-linked
with only the
subunit of RNA polymerase via the CRP
N-terminal 52-loop. After photoactivation in the presence of cAMP and
promoter DNA, a novel protein band with a molecular mass of
approximately 100 kDa was observed on the Western blot which
cross-reacted with both the anti-CRP (64D1) and anti-
(2D1)
monoclonal antibodies (Fig. 2). Several bands which cluster
around 45 kDa were also produced in the presence or absence of cAMP.
Since these are recognized only by anti-CRP antibodies and are produced
even in the absence of RNA polymerase (results not shown), we believe
they are cross-linked CRP monomers. No cross-linking of CRP with
,
, or
` was detected (results not shown).
Figure 2: Western blotting experiments showing the photo-cross-linking results using the BPM-derivatized mutant CRPs on CCpmelR promoter DNA fragment. The CCpmelR DNA is 186 bp containing the promoter region from -90 to +96; the nonspecific DNA is 242 bp containing the C-terminal sequence of the crp gene from +481 plus a small sequence of the adjacent pBR322 vector. The indicated DNA fragments were produced by PCR. A, BPM-CRP C178S,G56C was used for photo-cross-linking. B, BPM-CRP C178S,K52N,G56C was used for photo-cross-linking.
The
photo-cross-linking between the CRP 52-loop and subunit of the
RNA polymerase holoenzyme is cAMP-dependent in the presence of promoter
DNA. In the absence of cAMP, the cross-linked product of
100 kDa
was not detectable, whereas the cross-linked CRP dimers at
45 kDa
were still present. In the presence of cAMP and the class II promoter
DNA CCpmelR, both BPM-CRPs (C178S,G56C and C178S,K52N,G56C)
cross-linked with the
subunit (Fig. 2). The mutant
BPM-CRPs were able to cross-link with
on a class I promoter CC+20pmelR but with lower efficiency. The cross-linking
efficiencies of the CRP derivatives on the gal-like CCpmelR or lac-like CC+20pmelR with
are better than on the natural gal and lac promoters, presumably because of the consensus CRP binding
sites on these synthetic promoters (Fig. 3).
Figure 3: Western blotting experiment showing the photo-cross-linking results for BPM-C178S,K52N,G56C on different DNA promoters. The CCpmelR DNA is 186 bp containing the promoter region from -90 to +96; the CC+20pmelR DNA is 206 bp containing the promoter region from -110 to +96; the gal DNA is 209 bp containing the promoter region from -170 to +39; the lac DNA is 203 bp containing the promoter region from -140 to +63; The indicated DNA fragments were produced by PCR.
In order to
determine the specificity of the cross-linking of CRP 52-loop with
, another CRP mutant C178S,T158C was made with a
cysteine substitution at the 158-position in the surface-exposed loop
156-162 present at the C-terminal domain of CRP. This mutant CRP
retained about 60-70% of the stimulating activity on both gal and lac promoters in vivo compared to the wild
type (data not shown). The same UV cross-linking reaction was carried
out, and the results are shown in Fig. 4. C178S,T158C was found
to cross-link with the
subunit of RNA polymerase consistent with
previously published results (Chen et al., 1994), while no
cross-linking was detected between this mutant and the
subunit. Their results have shown that the 156-loop is close to
the RNA polymerase
subunit on the lac promoter. Our
results indicated that this loop can cross-link with
not only on lac but also on gal promoter and this cross-linking
is strictly DNA-dependent.
Figure 4: Western blotting experiment showing the photo-cross-linking results for BPM-C178S,T158C on gal and lac DNA promoters. The gal and lac DNA promoters were produced by PCR as before.
To strengthen the conclusion that the
cross-linking of CRP with relies on the functional
preinitiation complex, substrate nucleotides (ATP, CTP, UTP, and
OMeGTP) were incubated with the (BPM-(C178S,K52N,G56C))
gal P
RNA polymerase ternary complex mixture
before UV irradiation. After a 5-min incubation at 37 °C, a
10-nucleotide-long transcript (terminated by the presence of OMeGTP)
was allowed to form and then treated by UV irradiation as usual. The
results indicated that following initiation of transcription and the
clearance of the gal promoter, the cross-linked
CRP-
product was not produced to a significant
extent (Fig. 5).
Figure 5:
Western blotting experiment showing the
photo-cross-linking results for BPM-C178S,K52N,G56C with RNA polymerase
holoenzyme before or after the clearance of the gal DNA
promoter. The reaction conditions given under ``Materials and
Methods'' under Photo-cross-linking were used. A
nucleotide substrate mixture (final concentration of 0.4 mM
each ATP, CTP, UTP and 0.08 mM OMeGTP) was added to the
CRPgal P
RNA polymerase ternary
complex followed by incubation at 37 °C for 5 min before UV
irradiation.
Compelling evidence indicates that direct contact between RNA
polymerase and CRP is required for transcription activation. In the
case of Class I promoters such as lac P1 contact involves CRP
loop-161 and the C-terminal region of the subunit of RNA
polymerase. In Class I promoters, the DNA site for CRP is centered at
position -61.5 or further upstream (Ishihama, 1988). The
involvement of the
subunit in activation is not limited to CRP
but also includes a number of other transcription factors which bind to
Class I sites (Ishihama, 1993).
Class II promoters such as gal P1 (CRP binding site centered at position -41.5) are
affected to varying extents by CRP mutants which result in
loss of transcription activation from Class I promoters. West et
al.(1993) showed that amino acid substitution in the 159-contact
loop (H159L,T158A) reduced CRP-dependent activation according to the
Class II promoter used. Earlier work indicated that mutation in the
loop involving amino acid 52 could reactivate the CRP
mutation at position 159 (Bell et al., 1990). It was
suggested by West et al.(1993) that mutation in the 52-loop
could ``improve or create a second ``patch'' on
CRP'' which may contact RNA polymerase.
We have provided direct
evidence for contact between the 52-loop and the subunit of RNA
polymerase. Cross-linking is specific for the
subunit and
requires the cAMP-bound conformer of CRP. Little cross-linking occurs
in the absence of DNA. The mutants prepared for the present study show
activity comparable to wild type CRP for lac expression.
Mutant CRP C178S,K52N,G56C supports a 3-fold increased expression from
the gal-like promoter, CCpmelR (Fig. 1).
Replacing lysine 52 with asparagine (Bell et al., 1990) or
glutamic acid (West et al., 1993) had previously been shown to
stimulate expression from CCpmelR. Cross-linking carried out
in the presence of promoter DNA shows the discrimination between lac-like and gal-like promoters.
Transcription
factors can interact with RNA polymerase both in the presence and
absence of promoter DNA (Ishihama, 1988). The contact site of CRP with
RNA polymerase on Class I promoters has been located on the 159-loop of
CRP and the C terminus of the subunit of RNA polymerase. While
the C terminus of
is indispensable for factor activation on Class
I promoters, it does not appear to be essential for Class II promoter
activation. On Class II promoters like gal P1, where the CRP
binding site is located at the -41.5-position, it appears that
protein-protein contact also plays an important role in formation of
the CRP
RNA polymerase
DNA complex essential for
transcription initiation. Kumar et al.(1994) demonstrated that
deletions of the 4.2 conserved region of
responsible for -35 recognition could still effectively
transcribe gal P1 in the presence of cAMP-CRP. They also
proposed that the CRP contact site for gal P1 activation could
be located between amino acids 530 and 539 on
. Our
cross-linking results are consistent with their results in
demonstrating that CRP physically contacts
. Whether
the
region comprising amino acids 530-539
actually is the site for contact with CRP remains to be determined.
In order to demonstrate that the formation of a functional
preinitiation complex on the gal promoter is necessary for the
cross-linking between the CRP 52-loop and subunit
of RNA polymerase, photo-cross-linking assays were carried out after
the initiation of transcription. The ATP, CTP, UTP, and OMeGTP mixture
was added to the preinitiation complex as substrates for transcription.
Since the initial 10-ribonucleotide sequence of the gal P1
transcript is AUACCAUAAG . . . , this would enable transcription to
initiate but pause after incorporation of the first 10 ribonucleotides
because of the addition of the chain-terminating OMeGMP at the 10th
position. Following photoirradiation, cross-linking between CRP and
could hardly be detected after the initiation of
transcription. This result clearly demonstrated that CRP-
cross-linking relies on the formation of a functional
transcription complex.
The surface-exposed loop of CRP spanning
amino acids 156-162 has been shown to be in close contact with
the C terminus of the subunit of RNA polymerase on the lac promoter (Chen et al., 1994). To further establish the
specificity of the cross-linking of our N-terminal CRP 52-loop
mutations with the
subunit, a cysteine substitution
at the 158 position was made (C178S,T158C). This mutant was used for
cross-linking reactions using the same conditions as for the 52-loop
substitutions. The cross-linking results on the lac promoter
were the same as those reported by Chen et al.(1994);
C178S,T158C cross-linked with the
subunit of RNA polymerase on
the lac promoter, while no cross-linking with
,
`,
or
was detected. This cross-linking was also cAMP- and
DNA-dependent. Surprisingly, C178S,T158C also showed a similar
efficiency for cross-linking with the
subunit on gal as
well as lac promoters. Recently published evidence also
indicated that CRP contacts the
subunit of RNA polymerase on the gal P1 promoter as demonstrated by DNA footprinting (Attey et al. 1994). They suggested that
binds at the upstream
end of both the binary RNA polymerase
gal P1 and ternary
RNA polymerase
CRP
gal P1 complexes. In the ternary
complex,
appears to make direct contact with activating region 1
(which includes the 156-162-loop) in CRP. Taking this into
consideration, our results showing cross-linking of C178S,T158C with
on both lac and gal promoters not only
clarified the specificity of cross-linking (52-loop with
and
158-loop with
), but also provided direct evidence that the
activating region 1 of CRP makes direct contact with
on lac as well as on gal promoters.
No direct evidence is
available indicating that the 52-loop of CRP is essential for
stimulating transcription of gal P1. It remains possible that
the contact between the CRP 52-loop and is gratuitous and not
directly involved in transcription activation. It is also possible that
more than one site on CRP may contact one or more RNA polymerase
subunits for overall stimulating activity. So far, at least two of the
RNA polymerase contact sites on CRP have been identified. West et
al.(1993) suggest that neither the 52-loop nor the 159-loop is
essential for activating transcription on Class II promoters, another
activating region, perhaps located around glutamic acid 96, might also
be involved in transcription stimulation.