(Received for publication, June 6, 1995; and in revised form, August 23, 1995)
From the
Mutations conferring streptolydigin resistance onto Escherichia coli RNA polymerase have been found exclusively in
the subunit (Heisler, L. M., Suzuki, H., Landick, R., and Gross,
C. A.(1993) J. Biol. Chem. 268, 25369-25375). We report
here the isolation of a streptolydigin-resistant mutation in the E.
coli rpoC gene, encoding the
` subunit. The mutation is the
Phe
Ser substitution, which occurred in an
evolutionarily conserved segment of the
` subunit. The homologous
segment in the eukaryotic RNA polymerase II largest subunit harbors
mutations conferring
-amanitin resistance. Both streptolydigin and
-amanitin are inhibitors of transcription elongation. Thus, the
two antibiotics may inhibit transcription in their respective systems
by a similar mechanism, despite their very different chemical nature.
Streptolydigin (Stl) is a 3-acyltetramic acid
antibiotic(1) , which specifically inhibits bacterial
DNA-dependent RNA
polymerase(2, 3, 4, 5, 6) .
Stl interacts with RNAP in ternary transcription complexes and inhibits
growth of nascent RNA chains during transcription initiation and
elongation(4, 6) . The binding of RNAP to template DNA
is not affected by Stl(2) . Thus, the likely target of Stl is
either the binding of incoming NTP in the substrate binding site of
RNAP or the catalysis of phosphodiester bond
formation(4, 6) .
Transcription by RNAP purified
from mutant cells that acquired resistance to the drug is resistant to
Stl(3) . In Escherichia coli, all Stl-resistant RNAPs
studied to date have an altered subunit(7, 8) ,
and all known mutations leading to Stl
RNAP map to the rpoB gene, which codes for the
subunit(8) .
Substitutions of amino acids in
between 540 and 546 lead to Stl
resistance(9, 10, 11) . The highest
resistance levels in vivo and in vitro were found in
the case of substitutions at positions 544 and
545(10, 11) . In the absence of more direct data it
has been assumed that these
amino acids participate in Stl
binding to RNA polymerase.
Since Stl inhibits elongation of nascent
RNA, mutations changing RNAP Stl-binding site are likely to change the
catalytic properties of the enzyme. However, RNAP with the known
subunit Stl
mutations has unaltered transcription
elongation and transcription termination properties in vivo and in vitro(11) . Moreover, the site of Stl
resistance in the
subunit is dispensable for RNAP function, since
mutant RNAPs with deletions spanning amino acids 534-545 are
functional both in vivo and in vitro(10) .
This apparent discrepancy led Heisler et al.(11) to
hypothesize that other site(s) in RNAP may be involved in Stl binding.
This hypothesis was substantiated by a report (12) that in RNAP
reconstituted in vitro, the
` subunit from a mutant
strain of Bacillus subtilis was responsible for Stl
resistance. However, no
` subunit Stl
mutants have
ever been reported in E. coli. Here, we report an isolation
and localization of such a mutation. The mutation leads to an amino
acid substitution in an evolutionarily conserved region of the
`
subunit. In the
` homologues from eukaryotic RNA polymerase II,
this region harbors mutations that lead to resistance to
-amanitin, a peptide toxin that specifically inhibits RNA chain
elongation by RNA polymerase II. Our results raise the possibility that
the structurally different inhibitors streptolydigin and
-amanitin
may interact and inhibit RNA polymerase from prokaryotic and eukaryotic
systems by a similar mechanism.
Transcription
of bacteriophage T2 DNA was performed in 100-µl reactions
containing 10 µg of T2 DNA, 2 µg of WT or mutant RNAP, 0.5
mM ATP, CTP, and GTP, 0.025 mM [H]UTP (43 Ci/mM), 10 mM Tris-HCl (pH 7.9), 100 mM NaCl, 50 mM KCl, 10
mM MgCl
, 0.5 mg/ml bovine serum albumin, and
different concentrations of Stl. Reactions were initiated by addition
of NTPs and proceeded for 15 min at 37 °C. Reactions were
terminated by addition of 1 ml of 10% trichloroacetic acid, and the
amount of acid-insoluble radioactivity was determined.
To determine
the elongation rate of the mutant RNAP, elongation complexes stalled at
position +20 were prepared in the reactions containing (50
µl): 25 µl of Ni-NTA-agarose (Qiagen, Inc.),
20 nM of the 302-bp T7 A1 promoter DNA fragment (template 3 of
Nudler et al.; (39) ), 40 nM RNAP, 0.5
mM ApU, 50 µM CTP and GTP, 2.5 µM [
P]ATP (10 Bq/fmol), 40 mM Tris-HCl (pH 7.9), 40 mM KCl, 10 mM
MgCl
. Reactions proceeded for 15 min at 23 °C and were
washed three times with 1.5 ml of the buffer (40 mM Tris-HCl
(pH 7.9), 40 mM KCl, 10 mM MgCl
) as
described(13) . +20 elongation complexes were
synchronously started by making reactions 10 µM with NTPs.
Reactions proceeded for 0-120 s at 23 °C. Reactions were
terminated by addition of EDTA to 15 mM. To determine
transcription termination efficiencies of mutant enzymes, +20
elongation complexes were prepared as described above using 324-bp DNA
fragment (template 1 of Nudler et al.; (39) )
containing T7 A1 promoter followed by phage
tR2 terminator.
Transcription was started by addition of 1 mM NTPs. Reactions
proceeded for 2 min at 23 °C. Products were analyzed by
urea-polyacrylamide gel electrophoresis (7 M urea, 6%
polyacrylamide), followed by autoradiography and PhosphorImager
analysis.
The purpose of this study was to generate mutations in E.
coli rpoC that would result in Stl RNAP. E. coli cells are naturally resistant to high concentration of Stl, due to
a permeation barrier(2) . Mutant E. coli that become
sensitive to low concentration of Stl can be used for selection of
mutants with Stl-resistant RNAP(2, 11) . Throughout
this work, we used the Stl-sensitive E. coli strain CAG 14064,
provided by C. Gross. To obtain Stl
mutations, rpoC expression plasmid pMKa201 (13) was mutagenized with
hydroxylamine in vitro(11) . After mutagenesis,
plasmid DNA was introduced into CAG 14064 strain by electroporation and
cells were plated on media containing 12.5 µg/ml Stl. Out of
1
10
plasmid-bearing cells plated on the selective
media, 20 Stl
clones were obtained. Plasmid DNA was
prepared from the resistant clones and retransformed in CAG 14064, and
cells were plated on Stl-containing media. In this way two pMKa201
derivatives (pMKa201-15 and pMKa201-16) that conferred Stl resistance
to CAG 14064 strain were selected and used for further analysis.
To
localize Stl mutations, a series of in vitro exchanges of DNA fragments between the mutant plasmids pMKa201-15
and pMKa201-16, and the parental pMKa201 were performed. The
recombinant plasmids were checked for their ability to support growth
of CAG 14064 cells on 12.5 µg/ml Stl. In this way, the determinant
of the Stl
phenotype in both mutant plasmids was shown to
reside in the 997-bp SphI-SalI fragment of rpoC (Fig. 1A). The fragment was sequenced in both
orientations in pMKa201-15, pMKa201-16, and the parental pMKa201.
Comparison of the sequences revealed a single nucleotide difference
between pMKa201 and the mutant plasmids. Both mutant plasmids have a
TTC at codon position 793, while pMKa201 has a CTC at that codon
position, as does the published sequence(14) . As a result of
this change, the
` subunit expressed from the mutant plasmids has
a phenylalanine at amino acid position 793 instead of serine, as found
in the wild-type protein (Fig. 1B). Since we have
sequenced the entire fragment responsible for Stl resistance of
pMKa201-15 and pMKa201-16 and the CG to TA transition is the only
change from the wild-type sequence, we conclude that the change from
phenylalanine to serine at
` position 793 is the cause of
Stl
phenotype. We have named the Stl
mutation rpoCS793F and will refer to the mutation by that name.
Figure 1:
rpoC mutation S793F. A,
localization of rpoCS793F by transfer of the indicated
fragments from plasmid pMK201-15 into recipient parental plasmid
pMK201. Restriction sites used are indicated. The failure of
recombinant plasmids to confer Stl resistance upon the sensitive host
was taken as evidence that the transformed fragment did not carry the
Stl mutation. B, genetic context of rpoCS793F. The heavy bar represents the 1407 amino
acid
` subunit of E. coli RNAP. Hatched boxes labeled A-H represent segments of
` highly
conserved in evolution. The amino acid sequence of E. coli
` subunit conserved region F is expanded underneath.
Nucleotide sequence around codon 793 is shown in the box with
the mutant base set in lowercase. The five
termination-altering segment F mutations (15) that conferred
low levels of Stl resistance are shown above the E. coli sequence in italics. Homologous amino acid sequences from P. putida (P.p., (24) ), chloroplasts from
spinach (S.o., (25) ), Drosophila melanogaster Pol II (D.m., (26) ), and methanobacterium (M.t., (27) ) are also shown. The dots symbolize identity to the E. coli sequence, and the hyphens represent gaps. The known amino acids substitutions in
the Pol II largest subunits of mouse(28, 29) , Drosophila(30) , and C. elegans(31) leading to
-amanitin resistance are indicated.
Other
` features, including a surface-exposed putative zinc
finger(32) , a site where homologues from chloroplasts and
archaebacteria are split(33, 34) , a site where 3` end
of the nascent transcript cross-links to
` (35) , and a
site of a large (>200 amino acids) insertion in chloroplast
homologues(36) , are shown above the bar representing
`. A site of a large deletion in homologues from M. leprae(37) and T. maritima(38) and B.
subtilis (see Footnote 2) is shown by the open
box.
The
results of plating of CAG 14064 cells expressing rpoCS793F from the pMKa201 plasmid on a plate containing a linear gradient
of Stl are shown in Fig. 2A. Cells overproducing the
mutant ` subunit continued to grow at
20 µg/ml Stl, while
cells overproducing wild-type
` from the pMKa201 failed completely
to form colonies on the gradient plate.
Figure 2: A, in vivo levels of Stl resistance of E. coli expressing rpoCS793F. Panel shows overnight growth of cells on a plate containing a linear gradient of Stl concentration. B, transcription by S793F RNAP in the presence of Stl. WT and S793F RNAPs were purified from induced cell cultures as described(13) , and used to transcribe bacteriophage T2 DNA. Transcription activity in the presence of the indicated concentrations of Stl is presented as a percentage of the activity in the absence of Stl.
The ` subunit expressed
from the plasmid pMKa201 or its derivatives is extended with a stretch
of six consecutive histidine residues at its C terminus (the His tag).
As is shown elsewhere, the His-tagged RNAP is indistinguishable from
the wild-type RNAP in functional tests and can be easily separated from
RNAP with chromosome-encoded
` by affinity chromatography on
Ni
sorbent(13) . We purified His-tagged RNAP
from cells harboring pMK201 or pMK201-rpoCS793F.The response
of the two enzymes to Stl was compared in bacteriophage T2 DNA
transcription assay (Fig. 2B). The enzymes displayed
equal levels of activity in the absence of Stl (data not shown). In the
presence of Stl, the mutant enzyme was clearly more active than the
control WT enzyme (half-inhibition at 100 and 10 µg/ml Stl,
respectively).
Recently, one of us performed a systematic search for
termination-altering mutations in the cloned E. coli rpoC gene(15) . The mutations clustered in several regions of
the gene. Many of the termination-altering mutations resulted in amino
acid substitutions in a segment of the ` subunit between amino
acids 630 and 800 (interval 3, see (15) for nomenclature).
Since rpoCS793F is contained within interval 3, we
investigated the ability of interval 3 termination-altering mutations
to confer Stl
phenotype to Stl-sensitive cells. CAG 14064
cells harboring 19 pRW308 rpoC expression plasmid derivatives
carrying interval 3 mutations were streaked on plates with a linear
gradient of Stl. As a control, 8 interval 2 (amino acids 310-390)
and 10 interval 5 (amino acids 1305-1370) mutant plasmids were
used. Out of 37 plasmids tested, only 5 plasmids with interval 3
mutations (rpoC3302 (M747I), rpoC3309 (R780H), rpoC3310 (G729D), rpoC3312 (E756K), and rpoC3329 (M725I)) conferred very low levels of Stl resistance to CAG 14064
cells (Fig. 3A, and data not shown). rpoC alleles 3302 (M747I) and 3309 (R780H), which conferred higher
levels of resistance, were recloned in the pMKa201 plasmid; the two
His-tagged mutant RNAPs were purified, and their response to Stl was
investigated in the T2 DNA transcription system (Fig. 3B). In the absence of Stl, the mutant enzymes
were 50% more active than the control enzyme (data not shown). The two
enzymes reproducibly demonstrated slightly higher levels of Stl
resistance than the control enzyme (half-inhibition at 25 µg/ml
Stl).
Figure 3: A, in vivo levels of Stl resistance of E. coli expressing rpoCM747I and rpoCR780H. B, transcription by M747I and R780H RNAPs in the presence of Stl.
Since Stl inhibits phosphodiester bond formation, it is
expected that mutations in Stl-binding site will change the catalytic
properties of RNAP. Transcription elongation, transcription pausing,
and transcription termination by the three mutant enzymes (M747I,
R780H, and S793F) and WT RNAP were investigated in the experiment
presented in Fig. 4. The three mutant enzymes elongated RNA at
different rates (Fig. 4A); S793F RNAP was slightly
``slower'' than the WT enzyme, while M747I and R780H enzymes
were considerably ``faster,'' in agreement with the previous
data(15) . All three mutant enzymes and the WT control
demonstrated essentially the same pausing pattern in this assay.
Changes in transcription elongation rates were accompanied by changes
in transcription termination efficiencies of the mutant enzymes on a
factor-independent tR2 terminator (Fig. 4B);
S793F RNAP terminated slightly more efficiently (10% read-through),
while M747I and R780H enzymes terminated considerably less efficiently
(53 and 48% read-through, respectively) than the wild-type enzyme (20%
read-through).
Figure 4:
Transcription by mutant RNA polymerases. A, transcript elongation at undersaturating NTP concentration.
Elongation complexes stalled at position +20 of the T7 A1
transcription unit were prepared. Transcription was resumed by making
reactions 10 µM with NTPs. Reactions proceeded for the
times indicated, and reaction products were analyzed by denaturing
polyacrylamide electrophoresis followed by autoradiography. B,
transcription termination at -independent tR2 terminator of phage
. +20 elongation complexes were prepared on a T7 A1
transcription unit fused to the tR2 terminator. Transcription was
resumed by addition of 1 mM NTPs. Reactions proceeded for 2
min at 23 °C. Products were analyzed as in A.
The response of the mutant enzymes to other transcription inhibitors also was investigated. The mutant enzymes were as sensitive to initiation inhibitor rifampicin and elongation inhibitor tagetitoxin as the wild-type control (data not shown).
The principal result of this work is the demonstration that
mutations in the ` subunit of E. coli RNAP can confer
resistance to Stl. From the point of practical E. coli RNAP
genetics, the availability of an Stl resistance marker in rpoC should facilitate isolation of loss-of-function rpoC mutations, similarly to the approach that was used with rpoB (RNAP
subunit) mutations employing the rifampicin resistance
marker(16, 17) .
Biochemical analysis of the three
mutant RNAPs (M747I, R780H, and S793F) demonstrates that the extent of
defects in transcription elongation, transcription pausing, and
transcription termination of the mutants studied is not correlated with
the levels of Stl resistance. This situation is reminiscent of that for
the subunit Stl
mutants, which do not demonstrate
significant transcription defects(11) . We note that the S793F
mutation occurred in the highly conserved Segment F of
` (Fig. 1B), and that Stl
mutations affecting
RNAP basic function may have escaped our screen which requires mutant
RNAP in vivo function.
It is conceivable that additional
changes close to E. coli ` position 793 will be
identified that will lead to higher levels of streptolydigin
resistance. While this work was in progress, the B. subtilis Stl
rpoC mutation (12) was
sequenced(40) . The B. subtilis mutation projects on E. coli
` position 792, i.e. just next to S793F
mutation isolated in this study. Thus, the
` determinants of RNAP
streptolydigin resistance coincide in Gram-negative E. coli and Gram-positive B. subtilis.
Bacterial RNAP `
subunits are highly homologous to the large subunit of eukaryotic RNA
polymerase II(18) . As is shown in Fig. 1B, the
` Stl
mutations characterized in this work occurred in
a segment of
` that is highly conserved in evolution (Segment F,
according to (15) nomenclature). Several lines of evidence
suggest that this segment plays an important role conserved in all RNA
polymerases. Mutations in Segment F that dramatically change nascent
RNA elongation rate and/or termination efficiencies were reported in
eukaryotic as well as prokaryotic RNAPs (19, 15) . A
recent cross-linking study demonstrates that the E. coli
` segment between amino acids 748 and 814, containing most of
segment F, is in tight contact with the 3` end of the nascent RNA. (
)Finally, in eukaryotic RNA polymerase II largest subunit,
Segment F harbors mutations that render RNA polymerase resistant to the
elongation inhibitor
-amanitin (Fig. 1B). The
discovery of Stl
mutations in Segment F suggests that
despite the lack of structural similarity, Stl and
-amanitin may
inhibit transcription by a similar mechanism. Although speculative,
this hypothesis is consistent with available biochemical data on the
mechanism of Stl and
-amanitin inhibition of transcription. (i)
Both are elongation inhibitors, but allow several phosphodiester bonds
to be made and different complexes are inhibited to a different
extent(4, 20, 21) ; (ii) both inhibit
pyrophosphorolysis
(2) ; (iii) both inhibit
nascent RNA cleavage by transcription elongation
factors(22, 23) .