(Received for publication, May 23, 1995; and in revised form, August 30, 1995)
From the
Ribonucleotide analogs bound in the initiating site of Escherichia coli RNA polymerase-promoter complex were
cross-linked to the subunit. Using limited proteolysis and
chemical degradation, the cross-link was mapped to a segment of
between amino acids Val
and Arg
. This
region (Rif-cluster I) is known to harbor many rifampicin-resistant
(Rif
) mutations. The results demonstrate that Rif-cluster I
is part of the ``5`-face'' of the active center and provide
structural basis for the long-known effects of Rif
mutations on transcription initiation, elongation, and
termination.
The cellular multisubunit DNA-dependent RNA polymerase is the
central enzyme of gene expression and a target for genetic regulation.
The two largest subunits of RNA polymerase (RNAP) ()display
a remarkable degree of evolutionary
conservation(1, 2) . The best studied RNAP is that of Escherichia coli. The catalytically competent core RNAP
consists of the
` (1,407 amino acid residues), the
(1,342
residues), and the two
(329 residues) subunits(3) . The
holoenzyme composed of the core and a
subunit is able to
specifically initiate transcription from promoters(4) . The
initiation reaction begins with the binding of a priming substrate
(usually a purine nucleotide) and the next nucleoside triphosphate to
the holoenzyme stably anchored at the promoter DNA. RNAP then catalyzes
the formation of the first phosphodiester bond, yielding a dinucleotide
and pyrophosphate.
In order to identify amino acid residues
participating in the active center of RNAP, we took the approach of
mapping the sites of chemical cross-linking of substrate analogs. Using
probes specific for Lys and His residues, we showed that evolutionary
conserved Lys and His
near the C terminus
of the
subunit are located at the ``5` face'' of the
priming ATP(5, 6, 7) . Here, we use a broadly
specific alkylating cross-linkable group placed on the 5` side of the
initiating NTP to implicate another site of the
subunit in the
active center.
Three short regions in the middle of the
subunit defined by mutations of resistance to Rif, Rif-cluster I (amino
acids 512-534), Rif-cluster II (amino acids 563-574), and
Rif-cluster III (amino acid 687), have long been suspected of
involvement in the RNAP active center. The assumption rested on the Rif
ability to block extension of short initial transcripts(8) ,
the failure of Rif
mutants to bind Rif(9) , and
elongation defects observed in certain Rif
mutants (10, 11, 12) . However, all of these effects
could be explained by allosteric mechanisms whereby Rif
mutations and/or Rif would exert their effects at a distance.
Recently, we presented direct evidence positioning the Rif binding site
shortly upstream of the catalytic center (13) in accord with
the model that Rif plugs the product exit channel(8) . Yet,
there is still no rigorous proof that mutationally defined Rif-clusters
in the middle of
participate directly in Rif binding site or in
the RNAP active center. Our present results directly position
Rif-cluster I at the 5` face of the initiating NTP site.
Figure 4:
Localization of ATP*-cross-link site in
H
. A, the bar at the top represents the 1342-amino acid-long
-polypeptide, with lettered boxes symbolizing highly conserved sequence
regions(1) . The Rif region (10) and amino acids in the
C-terminal part of
that form the 5`-face of the active center (6, 7) are indicated. Underneath, successive steps of
chemical degradation of modified
H
are
schematized, corresponding to experimental data in B. The
cleavage sites that follow from
H
sequence are
shown by arrows. Radioactive
fragments are shown in black, the rest of the polypeptide is shaded. B,
H
RNAP was modified with ATP*. Purified recombinant
H
subunit was added (lanes 1 and 6), and complete CNBr degradation was performed (lanes 2 and 7). After affinity purification on Ni
sorbent (lanes 3 and 8), the bound material was
treated with BNPS-skatole (lanes 4 and 9), dried,
loaded on Ni
sorbent, and washed. The bound material
is analyzed in lanes 5 and 10. Reaction products were
resolved by electrophoresis in a 16% Tris-Tricine gel. The gel was
stained with Coomassie (left panel) and autoradiographed (right panel). In lane 6, radioactive bands migrating
faster than labeled
result from cross-linking to DNA (17) and disappear upon treatment with DNase (data not shown).
The faster migrating radioactive band in lanes 9 and 10 migrates slower than the corresponding Coomassie-stained band in lanes 4 and 5 due to covalently attached radioactive
dinucleotide tetraphosphate.
For Ni NTA agarose affinity purification, CNBr cleavage reactions were
dried down under vacuum and resuspended in 200-500 µl of
buffer containing 6 M urea and 100 mM Tris-HCl, pH
7.6. An aliquot was withdrawn for electrophoretic analysis, 25-50
µl of Ni
resin was washed in the urea buffer
added, and reactions were gently mixed for 30 min at 4 °C. After a
brief centrifugation, the sorbent was washed 3 times with 1.5 ml of the
urea buffer and then 2 times with 1.5 ml of transcription buffer. The
buffer was then removed and the bound protein was eluted in a small
volume of SDS-containing Laemmli loading buffer in the presence of 25
mM EDTA. For BNPS-skatole treatment, the affinity-purified
CNBr fragment was eluted from Ni
NTA agarose by
addition of 3 volumes of glacial acetic acid. 1 volume of BNPS-skatole
(10 mg/ml) in 70% acetic acid was added to the eluate, and reaction was
incubated for 10 h at room temperature, extracted to dryness with
ether, and the solid residue was dissolved in SDS-containing Laemmli
loading buffer.
For the high resolution mapping of the cross-links, we employ
genetically engineered catalytically proficient variants of RNAP with
conveniently located sites that facilitate analysis, i.e. a
highly labile proteolysis site about two-thirds down the length of the
subunit (15, 16) and an insertion of six
consecutive histidines between Rif-cluster I and Rif-cluster
II(19) . The inserted His-tag specifically absorbs to
Ni
-chelating sorbents and allows one to selectively
recover a subset of peptide fragments carrying the tag from a
proteolytic digest.
The adenine nucleotide analogs used in this work
as the primary cross-linkable reagents are shown on Fig. 1. The
AMP(1065) reagent was used in the previous
work(5, 6, 7) . Its aldehyde group
cross-links exclusively to the subunit
Lys
(7) . The three other reagents are analogous
to the previously described chloro derivatives(5) . The
reagents contain an alkylating group that is inactive because of a
strong electron-acceptor effect of the aldehyde group. Reduction of the
aldehyde group in the presence of NaBH
results in a
1000-fold increase of the alkylating activity. The iodo-containing
derivatives used in this work are
100 times more reactive than the
corresponding chloro compounds, and the alkylating reaction proceeds
with a half-life of 5 min at room temperature (data not shown). The
alkylating group can react with any nucleophilic amino acid side chain
(Cys, His, Lys, Tyr, Asp, Glu, Ser, Thr). However, alkylated Glu and
Asp residues are unstable and do not survive SDS-polyacrylamide gel
electrophoresis conditions.
Figure 1: Affinity labeling reagents. Structural formulae of derivatized adenine nucleotides used in this work are presented. The active groups are indicated in bold.
Figure 2:
Affinity labeling of RNA polymerase.
Affinity labeling of WT (A) and T989 (B) RNAP.
RNAP in the open complexes at the A1 promoter of phage T7 was
affinity-labeled with the reagents shown in Fig. 1. The
reactions were treated with trypsin as indicated and were then loaded
on an 8% Tris-glycine polyacrylamide SDS-gel. The cross-linked RNAP
subunits were visualized by autoradiography. In B, small
amounts of radioactive
fragment visible in the lanes
corresponding to reactions that were not treated with trypsin are due
to a very high sensitivity of
T989 RNAP to proteolytic attack
which occurs to some extent during the enzyme preparation. The
proteolyzed enzyme retains its catalytic activity (16) and is
therefore labeled in the assay.
When
small amounts of trypsin were added to completed modification
reactions, two new radioactive bands with apparent molecular size of
90 and
40 kDa appeared (lanes 2 and 3). In
the control experiment with AMP(1065) reagent, only the 40-kDa fragment
was seen in the trypsin-treated reactions (not shown). Elsewhere we
show that at the low concentrations used here trypsin predominantly
attacks WT RNAP at positions 903 and 909 of
, to generate two
fragments of
90 (N-terminal) and
40 (C-terminal)
kDa(15) . The results suggest that ATP* cross-linked both in
the C-terminal third and in the N-terminal two-thirds of the
-polypeptide.
Because only a small fraction of the WT RNAP is
cleaved under single-hit conditions, we repeated the above experiment
with a mutant enzyme carrying a highly labile proteolysis site
approximately two-thirds down the length of the -polypeptide. The
mutant
T989 RNAP is indistinguishable from the WT enzyme in
functional tests, but is much more sensitive to trypsin attack due to
an insertion of
130 amino acids at position
989(15, 16) . Fig. 2B shows the
results of an experiment where
T989 RNAP was modified with
different reagents and then subjected to mild treatment with trypsin.
When
T989 RNAP modified with the lysine-specific reagent
AMP(1065) (lane 1) was treated with trypsin, all of the
radioactivity was found in the C-terminal fragment of mutant
(lane 2). The alkylating cross-linkable group positioned at
the
-,
-, and
-phosphates of initiating adenosine
nucleotide, labeled both the smaller (C-terminal) and the larger
(N-terminal) fragments of
T989
(lanes 4, 6, and 8, respectively). In trypsin-treated affinity
labeling reactions, the part of the gel corresponding to intact large
subunits contained little or no radioactivity, testifying that
`
was not appreciably labeled by any of the reagents. Rifampicin
decreased the overall efficiency of labeling with ATP* reagent (lane 9), but did not inhibit cross-linking of the N-terminal
part of
(lane 10) (in fact, the ratio of the N- to
C-terminal fragment labeling increased in the presence of rifampicin).
Figure 3:
Complete CNBr degradation of cross-linked
N-terminal fragments. The radioactive
polypeptides shown in Fig. 2B were extracted from
the gel and treated with CNBr. Reaction products were separated on a
16% Tris-Tricine polyacrylamide-SDS gel, and radioactive peptides were
visualized by autoradiography.
Two major radioactive
peptides were generated when N-terminal fragments of T989
-labeled with alkylating reagents were subjected to CNBr
treatment. The larger peptide had an apparent molecular mass of
18
kDa, the smaller,
4 kDa. The ratio of the two peptides depended on
the affinity labeling reagent. The smaller peptide predominated when
AMP* was used (lane 1), while the larger was dominant when
RNAP was labeled with ATP* (lane 4). In the presence of Rif,
the amount of the smaller peptide cross-linked to ATP* decreased even
further (lane 5).
Since CNBr cleavage was complete, the
18-kDa labeled peptide should come from between two Met residues
separated by 140 amino acids. From the sequence of the N-terminal
tryptic fragment of
, the likely candidate is the fragment between
Met
and Met
. This was further tested using
a mutant
H
RNAP which carried a stretch of 8 amino
acids, Ile-(His)
-Trp, inserted between
positions 540
and 541(19) . The
H
RNAP is fully functional in vivo and in vitro(19) . Preliminary
experiments showed that the modified
H
enzyme gave
the same pattern of radioactive fragments upon trypsin cleavage as the
control WT RNAP (not shown). If the site of the ATP* cross-link in the
N-terminal part of
H
subunit were indeed
between Met
and Met
, we should be able to
purify the histidine-tagged, cross-linked
H
CNBr fragment by affinity chromatography on Ni
sorbent.
In the experiment of Fig. 4, the H
RNAP was affinity-labeled using ATP*, and an excess of unlabeled
H
subunit carrier was added so that the
material could be visualized both by staining (lane 1) and
autoradiography (lane 6). The CNBr reaction products shown (lanes 2 and 7) were loaded on Ni
NTA resin in the presence of 6 M urea and washed
extensively. As expected, CNBr-generated peptide
Val
-Met
was retained on the sorbent as
revealed by Coomassie staining (lane 3). Since the peptide was
radioactively labeled (lane 8), we conclude that the site of
the major ATP* cross-link in the N-terminal part of
is contained
within the fragment Val
-Met
. Control
experiments established that no polypeptides were retained on
Ni
NTA resin when WT RNAP was used in the
modification reaction (data not shown).
To further localize the site
of the ATP* cross-link, we made use of the Trp residue inserted
immediately past the six histidines in the H
RNAP.
The inserted Trp is unique to the Val
-Met
peptide. The affinity-purified Val
-Met
peptide was subjected to treatment with BNPS-skatole, a reagent
which specifically cleaves polypeptides after Trp residues. As is shown
on Fig. 4, lane 4, BNPS-skatole generated two peptides
with the apparent mobility of 5 and 11 kDa (expectant molecular sizes
are 3,678 Da and 12,850 Da, respectively). The smaller peptide was
radioactive, while the larger was not (Fig. 4, lane 9).
The larger BNPS-skatole peptide was lost during the second round of
affinity purification, while the smaller peptide as well as the initial
Val
-Met
peptide were retained on the
sorbent (lanes 5 and 10). We conclude that the site
of the ATP* cross-link is contained within the
subunit fragment
between Val
and Arg
.
The impetus for this work came from an earlier study (20) . ()The authors mapped a cross-linking site of
a 5`-chloro derivative of ATP between residues 516 and 653 of the WT
subunit. In the turmoil of disintegration of the Soviet Union,
these data were irreversibly lost, and we decided to reinvestigate the
issue using a more powerful family of iodo-derivatized reagents and
recombinant mutant RNA polymerases.
Our present results demonstrate
that the subunit segment between residues 516 and 540 contains
the site(s) of cross-linking to ATP*. This site should be located
within 5 Å of the
-phosphate of the priming ATP, which is
the effective range of the probe used. The distance between the
Rif-cluster I and the C-terminal
localities labeled by the same
reagent should not exceed this distance more than twice. Hence, these
two regions which are separated in the linear sequence of
by more
than 500 amino acids must be juxtaposed in the folded ternary structure
of the enzyme. Elsewhere we show that part of the conserved domain 3 of
, as well as positions -3 and -4 of the
template DNA strand are in contact with
-phosphate of initiating
nucleotide(17, 13) . In addition to these elements,
other RNAP sites apparently participate in the 5`-face of the active
center since another, yet unidentified site in the N-terminal part of
is efficiently cross-linked to ADP* and AMP* probes (see Fig. 3). Experiments aimed at mapping of these sites are in
progress.
Even though Rif-cluster I appears to be in direct contact
with the priming substrate, the cross-linked residue is not essential
for the enzymatic reaction since the derivatized enzyme is still able
to form the phosphodiester bond. The viability of many mutants isolated
in this area also argues against its direct involvement in an essential
function. On the other hand, the mutant RNA polymerases have been shown
to have altered apparent K values for the reaction
substrates during initiation and elongation(11) , as well as
changes in factor-dependent and independent
termination(10, 21) . These phenotypes are consistent
with the notion that the priming nucleotide site overlaps with a larger
site holding the RNA product in the active center during elongation.
Structural disruption of this area by a mutation may affect the
formation and/or proper positioning of the 3`-proximal portion of
nascent RNA and, hence, result in defects affecting extension of RNA
chains.
Our previous work positioned RNAP-bound Rif immediately
upstream of the initiating nucleotide binding site(13) . The
present results demonstrate that Rif-cluster I is located in the same
vicinity adding strength to the notion that Rif mutations
indeed define the Rif binding site.