From the Department of Microbiology, Ohio State
University, Columbus, Ohio 43210, the ¶ Laboratory of
Molecular Biophysics, Rockefeller University, New York, New York 10021, and the
Department of Bacteriology, University of Wisconsin,
Madison, Wisconsin 53706
Received for publication, November 3, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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The study of mutant enzymes can reveal important
details about the fundamental mechanism and regulation of RNA
polymerase, the central enzyme of gene expression. However, such
studies are complicated by the multisubunit structure of RNA polymerase
and by its indispensability for cell growth. Previously, mutant RNA polymerases have been produced by in vitro assembly from
isolated subunits or by in vivo assembly upon
overexpression of a single mutant subunit. Both approaches can fail if
the mutant subunit is toxic or incorrectly folded. Here we describe an
alternative strategy, co-overexpression and in vivo
assembly of RNA polymerase subunits, and apply this method to
characterize the role of sequence insertions present in the
Escherichia coli enzyme. We find that co-overexpression of
its subunits allows assembly of an RNA polymerase lacking a 188-amino
acid insertion in the The amino acid sequences of the two largest subunits of cellular
RNA polymerases (RNAPs),1
called In bacteria, the most studied examples of lineage-specific sequence
insertions (SIs)2 occur in
proteobacteria. In the proteobacteria, three easily recognizable
insertions protrude from the surface of the enzyme (Figs.
1 and 6), two in ' subunit. Based on experiments with this and
other mutant E. coli enzymes with precisely excised
sequence insertions, we report that the
' sequence insertion and, to
a lesser extent, an N-terminal
sequence insertion confer characteristic stability to the open initiation complex, frequency of
abortive initiation, and pausing during transcript elongation relative
to RNA polymerases, such as that from Bacillus subtilis, that lack the sequence insertions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
' and
in bacteria, are remarkably conserved among
multisubunit RNAPs from eubacteria to the human homologues, RNAPI,
RNAPII, and RNAPIII (1, 2). Recently determined crystal structures of
yeast and bacterial RNAPs (3, 4) reveal the structural congruity
responsible for this conservation, which extends far beyond the
catalytically important residues (5-7). Representing the bulk of the
enzyme,
' and
(and their yeast homologues, Rpb1 and Rpb2) form
two halves of a crab claw-shaped molecule, in which the secondary
structures of the homologous subunits are nearly identical within 25 Å of the active site (located at the internal junction between the
claws). The divergence among RNAP's large subunits generally increases
toward the exposed surface of the molecule and frequently is manifest
as insertions of up to several hundred amino acids that are
characteristic of different evolutionary lineages (3). This pattern of
surface sequence variation in RNAPs, which in eukaryotes includes
additional subunits bound at the enzyme's periphery, led to the idea
that the conserved catalytic core of RNAP is adapted to various
environments and cellular milieus by the addition of surface modules
that interact with different regulatory factors (4, 7, 8).
(SI1 between the
conserved regions B and C and SI2 between regions G and H) and one in
' (SI3 between regions G and H) (9, 10). These insertions are absent
in Gram-positive bacteria and in Deinococci, from which the
bacterial RNAP structures have been determined; the
Deinococci contain a different SI between conserved regions
A and B of
' (3, 11). Both
SI1 and
SI2 tolerate insertions and
partial deletions without affecting cell viability or in
vitro RNAP activities (9, 12, 13). In contrast, partial deletions
in
'SI3 are more deleterious; they reduce cell viability and confer
defects in transcript cleavage and elongation, whereas the complete
removal of
'SI3 inhibits assembly of the mutant
' into core RNAP
(14).
View larger version (23K):
[in a new window]
Fig. 1.
Linear maps of the '
and
subunits of RNAP. Conserved regions
of
' (A-H) and
(A-I) are shown in pink and
blue, respectively, with the structural domains of RNAP
indicated by the wider segments of the subunit maps and labeled using
the nomenclature of Darst and Kornberg (3, 4). SI indicates the
positions of amino acid sequence insertions in E. coli RNAP
that are not present in most bacterial species; respective boundaries
are shown above each SI. The endpoints of deletions studied
here are given at the ends of dotted
lines (numbers listed are the last deleted
residues). Black triangles marked with
letters (a-k) indicate positions of other
sequence insertions in various organisms. a (at
184),
178-aa insertion in Odontella sinensis chloroplast;
b (at
391), 25-aa insertion in Xanthomonas
campestris; c (at
774), 70-aa insertion in
Leptospira biflexa; d (at
1135), 69-aa
insertion in Wolbachia pipientis; e (at
'144),
290-aa insertion in Thermotoga maritima; f (at
'164), 115-aa insertion in Thermus aquaticus;
g (at
'204), 115-aa insertion in Aquifex
aeolicus; h (at
'618), 76-aa insertion in
Mycoplasma pulmonis; i (at
'869), 99-aa
insertion in T. maritima; j (at
'1161), 53-aa
insertion in A. aeolicus; k (at
' 1302), 68-aa
insertion in T. maritima. These sequence insertions
represent only a subset of a growing number of apparent
lineage-specific insertions intercalated between the conserved regions
of
and
' subunits in bacterial RNAPs.
The observations that SIs can tolerate significant structural
alterations without the loss of RNAP function led to them being described as dispensable (13-15). SIs in related bacteria exhibit somewhat greater variability than the sequences of full-length and
' (e.g. SIs are ~70% conserved between
Escherichia coli and Hemophilus influenzae and
~60% conserved between E. coli and Shewanella
violacea, whereas the remaining parts of
and
' are ~85 and ~80% conserved between the two pairs, respectively).
Nonetheless, their retention in distinct bacteria with significant
sequence conservation suggests that they play some functional role. One role could be the recruitment of cellular regulatory proteins. For
instance,
SI1 appears to be the target of the phage T4 Alc termination factor (15). SI modules could also affect biochemical properties of RNAP more directly; both
'SI3 and
SI1 are proposed to make downstream DNA contacts that explain the greater stability and
longer downstream footprints of E. coli RNAP open complexes (relative to, for instance, Bacillus subtilis RNAP) (7, 16).
We became interested in the sequence insertions in proteobacterial RNAP
as possible explanations for the inability of B. subtilis RNAP to recognize hairpin-dependent pause signals as well
as the decreased open complex longevity and decreased abortive
initiation of B. subtilis RNAP (17). To facilitate study of
RNAPs with precise SI deletions, we developed a polycistronic
co-overexpression system for E. coli RNAP that relies on T7
RNAP-dependent transcription of all three core RNAP subunit
genes (rpoA, rpoB, and rpoC) on a
single plasmid. To facilitate purification of the recombinant RNAPs, we
appended an intein-chitin-binding protein (CBP) module to the ' C
terminus and a hexahistidine-hemagglutinin tag to the N terminus of
. This system allowed us to purify and test precise deletions of all
three SIs in E. coli RNAP and provides a generally useful
method for the study of mutant E. coli RNAP enzymes that
avoids the loss of activity and assembly problems that sometimes
arise with in vitro reconstitution methods (18, 19).
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EXPERIMENTAL PROCEDURES |
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Reagents and Proteins--
Oligonucleotides (listed in Table
I) were obtained from Operon Technologies (Alameda, CA); dNTPs
were from U.S. Biochemical Corp.; NTPs were from Amersham Biosciences;
[-32P]CTP was from PerkinElmer Life Sciences; and
other chemicals were from Sigma. Restriction and modification enzymes
were obtained from New England Biolabs. Linear DNA templates for
in vitro transcription were generated by PCR amplification
and purified using reagents (cat. A7170) from Promega (Madison, WI).
Construction of Deletion Mutants--
To delete SI1, an
rpoB fragment was PCR-amplified from pRL702 with
oligonucleotides 343 and 3135. The PCR product was digested with
NruI and cloned between two NruI sites of pRL702.
To delete
SI2 and
'SI3, site-directed PCR mutagenesis with two
fully complementary oligonucleotides flanking the deleted fragment in
pIA160 (
) or pRL663 (
') was performed. Each oligonucleotide (see
Table I) annealed on both sides of the deletion, forcing the
intervening fragment to loop out. The shortened rpoB
fragment located between the unique NcoI and ClaI
sites was sequenced, excised, and recloned into NcoI,
ClaI-cut pIA160 (pIA319) or NcoI, and
ClaI-cut pIA178 (pIA302). The shortened rpoC
fragment located between the unique SalI and
BspEI sites was sequenced, excised, and recloned into SalI-, BspEI-cut pRL663. To obtain overexpression
plasmids encoding
SI1 and SI2, the mutant rpoB
fragments were transferred to the T7 RNAP-based expression plasmid
pIA423 (Fig. 2) on an NcoI to
SdaI fragment. To obtain overexpression plasmids encoding
'
(943-1130), the mutant rpoC fragments were
transferred to the same expression plasmid on a BsmI to
XhoI fragment.
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Design of the Polycistronic Overexpression Plasmid--
The
original plasmid for co-overexpression of E. coli RNAP
subunit genes, pIA423 (Fig. 2; GenBankTM accession number
AF533984), contains a polycistronic operon rpoA-rpoB-rpoC*,
flanked by a single T7 promoter and terminator sequences derived from
pET21 (Novagen, Madison, WI); each ORF is preceded by a separate
ribosome-binding site (Fig. 2). Introduction of a
XhoI site at the 3'-end of the rpoC ORF resulted
in the addition of two amino acids (LE) to the C terminus of the '
subunit, whereas a new EagI site in rpoB ORF is
silent. In the absence of induction, expression is repressed by the
product of the lacIq gene (carried on the same
plasmid) through lacO positioned upstream of
rpoA. To facilitate purification of the recombinant RNAP,
the rpoC* fusion contains a CBP-intein module from pTYB3
plasmid (New England Biolabs) fused in-frame to the 3'-end of the
rpoC ORF. Derivatives of pIA423 containing hexahistidine and
a hemagglutinin epitope tag at the N terminus of
were prepared by
ligation of NcoI-SdaI fragments from pIA160 and
pIA178 (rpoBSF531) into the corresponding sites
of pIA423.
The co-overexpression plasmid pIA423 was created in the following steps
(see also Table I). pET21 was constructed to express rpoA
under the control of an IPTG-inducible T7 RNAP promoter. The
HindIII site in the pET21
rpoA gene was
eliminated without altering the encoded amino acid sequence by
site-directed PCR mutagenesis with primers 3683 and 3684 to yield
pIA287. pIA287 was converted to pIA299 first by introducing a new
sequence between the BamHI and NcoI sites
downstream of rpoA. The resulting plasmid was then modified
by insertion between its NcoI and HindIII sites of an NcoI to SbfI fragment from pRW408
carrying most of rpoB, followed by an SbfI
to BsmI fragment from pNF1346, carrying the remainder of
rpoB, the rpoBC intergenic region, and the first half of rpoC, followed by a BsmI to
HindIII fragment from pRL663, carrying the remainder of
rpoC, a C-terminal XhoI site, and a His6 tag. pIA423 was then created by insertion between
XhoI sites of pIA299 of a
XhoI-SalI-digested PCR product from pTYB3
(amplified with primers 3741 and 3742; Table I).
RNAP Purification--
Plasmids encoding variants of E. coli RNAP (wild type and mutants) were transformed into BL21
DE3 (20). A single colony was inoculated into 500 ml to 2 liters of
LB + 100 µg of ampicillin/ml at 37 °C and grown until apparent
A600 reached 0.3-0.5, at which point protein
production was induced by the addition of IPTG to 1 mM.
Cells were grown for 3 h at 37 °C, rapidly chilled on ice, collected by centrifugation for 15 min at 5000 × g and
4 °C, and resuspended in 50 ml of column buffer (20 mM
Tris, pH 7.9, 5% glycerol, 500 mM NaCl, 1 mM
EDTA). Protease inhibitor mixture (Sigma catalog no. P8465) was then
added as recommended by the manufacturer, and the cells were disrupted
by sonication. The resulting lysate was cleared by centrifugation for
20 min at 27,000 × g and 4 °C and then filtered
through the 0.4-µm syringe filter (Nalgene). Chitin beads (5 ml; New
England Biolabs) were equilibrated with 10 volumes of column buffer in
a 20-ml disposable column (Bio-Rad Econo-Pac), and cleared lysate was
passed through the column by gravity flow, followed by 20 volumes of
column buffer. To induce intein cleavage, the column was washed with 3 bed volumes of column buffer containing 50 mM DTT (to
exchange buffer) and then incubated at 4-8 °C for 8-16 h
(overnight). To elute the protein, column buffer (~4 ml) was added,
and 0.2-ml fractions were then collected and tested for protein by
Bradford assay and SDS-PAGE (4-12% NuPAGE gels; Invitrogen).
Fractions containing RNAP were pooled, concentrated using Centriplus
100 or Ultrafree concentrators (Millipore Corp.) to 1-5 ml (depending
on total RNAP recovered), and then dialyzed against loading buffer for heparin affinity or anion exchange chromatography. Chromatography was
carried out using HiTrap columns and an Akta Prime low pressure chromatography system (Amersham Biosciences). For heparin affinity separation, samples were loaded onto the HiTrap Heparin HP column in 50 mM sodium phosphate (pH 6.9), 0.1 mM DTT
buffer. For quaternary amine chromatography (Hi-Trap Q Sepharose Fast
Flow), protein was loaded in 50 mM Tris-HCl (pH 8.0), 5%
glycerol, 0.1 mM Na-EDTA, 0.1 mM DTT. Columns
were washed with 10 column volumes of the loading buffer and eluted
with a gradient (0-1.5 M) of NaCl in loading buffer over
20 column volumes. Elution peaks were identified by monitoring
A280, and their contents were further
characterized using SDS-PAGE. Fractions containing RNAP were pooled and
dialyzed against storage buffer (10 mM Tris, pH 7.9, 50%
glycerol, 0.1 mM EDTA, 0.1 mM DTT, 0.1 M NaCl) for 12-14 h at 4 °C. Enzymes containing
deletions in the
subunit were additionally purified by adsorption
to Ni2+-nitrilotriacetic acid beads (Qiagen), followed by
washing and imidazole elution prior to dialysis against storage buffer.
Typical yields from 2-liter cultures were 0.5-2 mg of purified RNAP,
depending on properties of the particular enzyme. For the wild type
RNAP, versions containing or lacking hexahistidine tags on
were
purified and found to behave indistinguishably in the assays used here. B. subtilis core RNAP (21) and
70 (22) were
purified as described. Wild type and mutant RNAP holoenzyme (core
'
2 plus
70) was prepared by
incubating a 5-fold molar excess of
70 with core enzyme
for 30 min at 30 °C.
Open Complex Longevity Assays--
Linear DNA template (40 nM) carrying the T7A1 promoter (pIA171) (23) was incubated
with 50 nM RNAP holoenzyme (with 70) for 15 min at 37 °C in 50 µl of transcription buffer (20 mM Tris·HCl, 20 mM NaCl, 10 mM
MgCl2, 14 mM 2-mercaptoethanol, 0.1 mM EDTA, pH 7.9). Heparin was added to 14 µg/ml to
sequester any free RNAP (at time 0); reaction aliquots were withdrawn
at various times and combined with nucleotide substrates (ApU to 150 µM; ATP, GTP, and CTP to 2.5 µM; and 5 µCi of [
-32P]CTP). Following a 10-min incubation at
37 °C, reactions were quenched by the addition of an equal volume of
STOP buffer (10 M urea, 20 mM EDTA, 45 mM Tris borate, pH 8.3). For the zero time point, an
aliquot was taken prior to heparin addition and incubated with substrates.
Abortive Initiation Assays--
Reactions were assembled on ice
in 50 µl of transcription buffer with ApU at 150 µM,
ATP and CTP at 20 µM, and 10 µCi of
[-32P]CTP (3000 Ci/mmol). Linear pRL550 (24) DNA
template was at 40 nM, and RNAP holoenzyme containing
70 was at 50 nM. Transcription was initiated
by shifting samples to 37 °C. Samples (5 µl) were removed at times
indicated in the legend to Fig.
3B and after a final 5-min
incubation with 500 µM each NTP (chase) and quenched by
the addition of STOP buffer.
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Single Round Pause Assays--
Linear DNA template encoding the
his pause signal was generated by PCR amplification of
pIA171 (23). Halted A29 elongation complexes were formed during a
15-min incubation of 40 nM DNA template and 50 nM RNAP holoenzyme at 37 °C in 50 µl of transcription buffer in the absence of UTP, with ApU at 150 µM, ATP and
GTP at 2.5 µM, and CTP at 1 µM, with
32P derived from [-32P]CTP (3000 Ci/mmol).
Transcription was restarted by the addition of 20 µM GTP;
150 µM ATP, UTP, and CTP; and heparin to 100 µg/ml. Samples were removed at the times listed in the figure legends and
after a final 5-min incubation with 250 µM each NTP
(chase) and were quenched as above.
Sample Analysis--
Samples were heated for 2 min at 90 °C
and separated by electrophoresis in denaturing acrylamide (19:1) gels
(7 M urea, 0.5× TBE; 8% for pause assays, 15% for
abortive initiation and open complex stability assays). RNA products
were visualized and quantified using a PhosphorImager and ImageQuant
Software (Amersham Biosciences). Pause half-life (the time during which
half of the complexes reenter the elongation pathway) was determined by
nonlinear regression analysis as described previously (25).
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RESULTS |
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Co-overexpression of RNAP Subunits Allows Study of the Role
of Proteobacterial Sequence Insertions--
To allow facile
expression, assembly, and purification of poorly assembled or toxic
mutant RNAPs, such as '
SI3, we designed a vector that expresses
,
, and
' polypeptides from a single T7 promoter; the
'
subunit is fused to the CBP tag (see "Experimental Procedures"). We
predicted that expressing the mutant core RNAP from this plasmid would
facilitate its assembly in vivo and would allow purification
via the CBP tag (Fig. 2). This approach has several advantages. First,
only the fully assembled core (
2
') RNAP is
purified because
' recruitment is the last step in the assembly
pathway (26) and because the expression levels from this vector follow
the assembly pathway of RNAP (
>
>
'; data not shown). Second,
intein-mediated cleavage removes the CBP tag to release the assembled
RNAP from the matrix; therefore, the purified enzyme does not carry
additional protein segments. Third, the entire purification can be
completed quickly, is relatively inexpensive, and yields RNAP that is
sufficiently pure for in vitro transcription (Fig. 2).
Fourth, the plasmid-encoded
and
' subunits assemble
preferentially with each other (and not with the chromosomal subunits;
data not shown), allowing combination of substitutions in different
subunits and more homogenous population of purified RNAPs. To purify
RNAP with altered
subunit, we also constructed a version of the
co-overexpression plasmid with a hexahistidine tag at the N terminus of
(see Table I).
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Using the co-overexpression plasmid, we were able to obtain active
'SI3 RNAP (Fig. 2) as well as RNAPs with precise excisions of
SI1 or
SI2. We found that RNAP eluted directly from chitin beads
was suitable for in vitro transcription but sometimes
exhibited reduced activity relative to RNAP purified by conventional
methods (27). We hypothesized that this low activity arose from
residual binding of nucleic acids to the RNAPs; the addition of one
chromatography step on heparin- or quarternary amine-Sepharose yielded
overexpressed RNAP of an activity and purity comparable with that
obtained by conventional purification (Fig. 2 and data not shown).
When the '
SI3-intein-CBP fusion was expressed from a T7 promoter
plasmid that did not encode the other RNAP subunits, essentially all
tagged
' was found in inclusion bodies (data not shown). Therefore,
co-overexpression with
,
, and
' subunits facilitates RNAP
assembly. Perhaps translation of
and
' from the same mRNA facilitates proper interaction, or the elevated level of the initially formed
2
complex simply allows plasmid-encoded
'
to compete effectively with chromosomally encoded
' for assembly.
'SI3 Stabilizes Open Initiation Complexes--
Using RNAPs with
precise SI deletions obtained by co-overexpression, we first tested the
effects of the E. coli SIs on open initiation complexes.
Fully mature E. coli open complexes, which form on many but
not all cellular promoters, exhibit extended contacts of RNAP with DNA
from ~
55 to ~+20 relative to the transcription start site and
melting of the DNA duplex between ~
12 and ~+2. These E. coli open complexes are long lived and collapse back to closed
complexes at rates of <0.01 s
1 (28-31). In contrast,
B. subtilis RNAP, which lacks insertion sequences, forms
open complexes that are in rapid equilibrium with closed promoter
complexes (32, 33). In B. subtilis RNAP open complexes, the
DNA downstream of the start site is not efficiently protected against
DNase I digestion (32, 34), and the melted region is shortened in the
downstream direction (35). Interestingly, a deletion variant of the
E. coli
subunit that includes part of
SI1 but extends
beyond its boundaries (
186-433) forms open complexes that (at
37 °C) exhibit a melting pattern similar to those formed by the
B. subtilis enzyme (35).
To measure the relative stability of open complexes formed by wild type
or SI RNAPs, we challenged open complexes formed on the T7 A1
promoter with the polyanion heparin. Although for many promoters
heparin binds only free RNAP that is in equilibrium with closed
initiation complexes and has no effect on open complexes, heparin
directly attacks open complexes formed at the T7 A1 promoter and
displaces RNAP from the promoter DNA in addition to binding free RNAP
(36). Upon heparin addition to 14 µg/ml, transcriptionally competent
open complexes (as assayed by the addition of NTPs and formation of an
RNA transcript at different time intervals following heparin challenge)
disappeared at a pseudo-first order rate of 0.006 s
1 for
wild type and
SI2 RNAP, about twice as fast for
SI1 RNAP, and about 10 times faster for
'SI3 RNAP and B. subtilis RNAP (Fig. 3A). We conclude that the
characteristic instability of B. subtilis open complexes is
mimicked by
'SI3 E. coli RNAP and that
SI1, but not
SI2, also contributes to the stability of E. coli open complexes.
'SI3 Promotes Abortive Initiation--
A second property that
distinguishes B. subtilis and E. coli RNAPs is an
ability of B. subtilis RNAP to escape promoters at which E. coli RNAP is trapped in abortive synthesis. We
found this previously using a variant of the T7 A1 promoter whose
initially transcribed sequence (5'-AUCCCACACC ...
versus wild type 5'-AUCGAGAGGG ... ) appears to act
cooperatively with strong contacts of E. coli RNAP to
promoter DNA to trap the enzyme in abortive synthesis (24); B. subtilis RNAP is able to escape from the mutant T7 A1 promoter
with relatively few abortive products made, and the patterns of
abortive products also differ between the B. subtilis and
E. coli enzymes (17).
To investigate whether any of the sequence insertions in E. coli RNAP might explain its characteristically poor escape from the mutant T7 A1 promoter, we measured the abortive to productive RNA
product ratios for the various RNAPs on this promoter (Fig. 3B). Interestingly, deletion of 'SI3 reduced the abortive
to productive product ratio, although only to a level still ~2-fold greater than found for B. subtilis RNAP. However, deletion
of either SI in
dramatically increased the ratio. Thus, the
E. coli sequence insertions profoundly affect promoter
escape, although in different directions;
SI1 and
SI2 promote
escape, whereas
'SI3 inhibits escape.
The patterns of abortive products produced also are revealing (Fig.
3B). Both SI1 and
SI2 RNAPs produce an altered
pattern of abortive products relative to wild type or
'
SI3 RNAPs
(compare the 60-min lanes for each RNAP in Fig.
3B). However, although
'
SI3 RNAP exhibited increased
promoter escape, like B. subtilis RNAP, it does not
recapitulate the distinctive pattern of B. subtilis abortive
products. We conclude that the absence of the Proteobacterial sequence
insertions substantially, although not completely, accounts for the
distinctive initiation properties of B. subtilis RNAP.
'SI3 Slows Escape from Pause Sites--
Another key difference
between the E. coli and B. subtilis RNAPs is
their responses to certain hairpin-dependent pause sites. Both enzymes can recognize hairpin-independent pause signals as well as
a B. subtilis P RNA pause site at which a nascent RNA hairpin 12 nt upstream from the pause is important in the presence, but
not in the absence, of NusA (17). However, B. subtilis RNAP fails to recognize the his pause
site, at which a nascent RNA hairpin 11 nt upstream from the pause site
contributes a factor of 5-10 to the delay in pause escape through an
interaction with the
flap domain of the E. coli RNAP
(17, 37).
The structural basis for this difference is not known; B. subtilis RNAP might be unable to respond to the RNA hairpin
formation or to other components of this signal (the downstream DNA,
the 3'-proximal RNA, and the nucleotides in the active site) that also
slow pause escape independently of the RNA hairpin. We previously found
that replacement of the E. coli flap with the
corresponding fragment of the B. subtilis
did not alter
pausing (17), suggesting that the difference in their response to the
his pause signal lay elsewhere. Thus, the sequence
insertions in E. coli RNAP were the next logical possibility.
To ask if the E. coli sequence insertions play a role in
pause site recognition, we first tested pausing on the his
pause template. We found that, unlike B. subtilis RNAP,
SI1,
SI2, and
'SI3 RNAPs all recognized the
his pause site (Fig. 4).
However,
'SI3 RNAPs escaped from the pause site ~5 times faster
than wild type and exhibited an ~2-fold decrease in the efficiency of
pause site recognition. To ask if the residual pausing by
'SI3 RNAPs was still hairpin-dependent, we tested the effect of
an antisense oligonucleotide that pairs to nascent pause RNA including the two 5'-most nt of the pause hairpin stem (Fig. 4A) and
that eliminates the 5-10-fold contribution of the hairpin to pausing by disrupting its structure (23). If
'SI3 somehow mediated the
effect of the pause hairpin, despite their locations on opposite sides
of the paused TEC, we would expect the antisense oligonucleotide to
have little effect on pausing by
'SI3 RNAP. However, the antisense oligonucleotide reduced the pause half-life of
'SI3 RNAP by a factor of ~3 (Fig. 4D), suggesting that
'SI3
contributes to pausing through interactions that are largely
independent of the hairpin interaction. We verified this conclusion by
testing the effect of
'SI3 on pausing at the hairpin-independent
ops pause site, where the deletion had effects on half-life
and efficiency nearly identical to the effects observed at the
his pause site (Fig. 5).
However,
'SI3 did not fully recapitulate the pausing behavior of
B. subtilis RNAP, which bypasses the his
pause site without apparent delay (Fig. 4B) and transcribes
through the ops pause site even more rapidly than does
'SI3 RNAP (17).
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We conclude that 'SI3 plays a central role in the strong pausing
behavior of E. coli RNAP but that differences between
E. coli and B. subtilis RNAP in addition to
'SI3 must contribute to reduced pausing by the latter enzyme.
Interestingly, a partial deletion in
'SI3 (
1091-1130) displays
the opposite effect on pausing as the precise
'SI3 deletion that we
studied; i.e. it increases rather than decreases pausing
(14). We return to the implications of this discrepancy under
"Discussion."
Multiple Deletions of E. coli RNAP Sequence Insertions--
Given
the distinct effects of SI1 and
'SI3 on open complex longevity,
abortive initiation, and pausing, we wondered how these effects would
combine in an enzyme lacking both SIs. All attempts to produce such an
enzyme failed, however (data not shown). Derivatives of pIA423 that
expressed
SI1 and
'
SI3 never yielded assembled RNAPs
bearing the deletions, even when we included on the co-expression
plasmid rpoZ, the gene encoding the dispensable RNAP subunit
that is reported to promote RNAP assembly (38).
Deletions of the Sequence Insertions Impair Growth of
Bacteria--
Finally, we wished to test the dispensability of
sequence insertions in RNAP for bacterial growth. Studies conducted
prior to the availability of high resolution RNAP structures on RNAPs lacking portions of SI1,
SI2, or
'SI3 led to the idea that these were dispensable regions, at least for core RNAP functions (9,
12-14, 39). Now that we could define precise deletions of the sequence
insertions and express functional enzymes containing these deletions
in vivo, we wished to revisit this idea and ask to what
extent the sequence insertions are required for bacterial growth.
To test the requirement for sequence insertions for bacterial growth,
we expressed the wild type or SI subunits from plasmids on which
their expression either singly or in combination with the other RNAP
subunits could be regulated by IPTG and lac repressor encoded on the same plasmids (Table I). We transformed the plasmids into strains that express wild type RNAP from the chromosome (rich medium, wild type subunit from chromosome expressed) (Table
II) or in which expression of the
corresponding subunit from the chromosome could be inactivated at 39 or
42 °C (for rpoC and rpoB, respectively; wild
type subunit from chromosome not expressed, Table II).
|
We found that plasmid-encoded expression of mutant subunit lacking
SI1 or SI2 in the presence of wild type
encoded on the chromosome
had little effect on cell growth However,
SI1 was unable to
substitute for wild type
at 42 °C, and it compromised growth at
30 °C when it contained a rifampicin-resistant amino acid
substitution (SF531) not present in the chromosomal copy of
rpoB and when rifampicin was added to the medium
(Table II). Growth on rifampicin at 30 °C was not restored when
SF531
SI1 was co-expressed with
' and
(conditions that yielded assembled
SI1 for purification). Thus,
SI1 appears to be incorporated into RNAP but unable to support
cell growth.
SI2 was able to support growth on rich medium even
at 42 °C or in the presence of rifampicin at 30 °C when combined
with SF531. However,
SF531
SI2 was unable to support
growth on minimal medium containing rifampicin, when expressed either
singly or together with
and
'. Thus, both sequence insertions in
E. coli
appear necessary for growth on minimal medium,
suggesting that they play some regulatory role in RNAP function that
becomes essential in stringent growth conditions.
We found that 'SI3 also was required for growth in some conditions;
however, the requirements for this sequence insertion are complex. A
'
SI3 plasmid could not be transformed into RL602 (Table I) in
which expression of chromosomally encoded
' is temperature-sensitive, even in the absence of IPTG (probably because low, uninduced expression of
'
SI3 produced too much defective enzyme when combined with the lowered expression of
' in RL602). The
'
SI3 co-expression plasmid could be transformed into the conditional rpoC strain but blocked growth when induced at
the permissive temperature and failed to support growth at the
nonpermissive temperature. Although induction of
'
SI3 in a strain
producing wild type
' lowered plating efficiency by 102,
it actually raised plating efficiency by >103 relative to
wild type
', when plated in the presence of an antibiotic (microcin
J25) that inhibits RNAP upon binding at a site near the
'SI3 in the
RNAP secondary channel (62). Co-expression
'
SI3 with
and
was not inhibitory in a strain expressing wild type RNAP (Table I) and
conferred nearly 100% plating efficiency in the presence of microcin
J25 (62). However, the growth rate of these colonies in the presence of
microcin J25 is drastically slower than strains carrying the
prototypical microcin J25 resistance mutation
(rpoCT931I). On balance, it appears that
'SI3
is essential for normal growth of E. coli but that, at least
in the presence of microcin J25, RNAP lacking
'SI3 can still support
slow growth on rich medium.
We conclude that sequence insertions, although dispensable for basic
RNAP function at all stages of the transcription cycle, nonetheless are
important to growth of the bacterium. This may reflect effects of the
sequence insertions on the basic steps in transcription by RNAP that
become crucial for expression of certain genes or in some growth
conditions but could also reflect roles in yet undescribed but
essential interactions of transcription factors similar to the
previously described interaction of the T4 Alc protein with SI1
(15).
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DISCUSSION |
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---|
We have described a co-overexpression system for mutant
E. coli RNAPs and its use to investigate whether
sequence insertions in the and
' subunits could explain the
different initiation and elongation properties of E. coli
RNAP compared with B. subtilis RNAP, which lacks these
insertions. We found that co-overexpression facilitates assembly of
otherwise difficult to assemble or toxic mutant RNAPs and that the
sequence insertions, most notably
'SI3, confer some of its
distinctive biochemical properties on E. coli RNAP and
partially account for its differences from B. subtilis RNAP.
We will discuss the implications of these findings for the study of
mutant RNAPs, the function of the E. coli sequence
insertions with focus on
'SI3, and the evolution of sequence
insertions in RNAP.
An RNAP Overexpression System That Promotes Efficient
Assembly--
Previous studies of toxic RNAP mutants have relied
either on conditional expression of a tagged mutant subunit followed by tag affinity separation from wild type RNAP (or tagging and removal of
the wild type enzyme from the RNAP preparation) (40) or in vitro reconstitution of RNAP by gradual renaturation of mixtures of the denatured subunits (19, 41-43). '
SI3 illustrates the limitation of these approaches. When expressed as an individual subunit
in vivo,
'
SI3 fails to out-compete wild type
' for assembly into useful amounts of mutant enzyme; when used in in vitro reconstitution protocols, it fails to assemble into RNAP (see "Results" and Ref. 14).
Co-overexpression of wild type and mutant tagged subunits solved these
problems for '
SI3 and has allowed us to isolate substantial quantities of many mutant RNAPs, including notably toxic mutants such
as the
(900-909) flap mutant RNAP (37). Thus, the
co-overexpression plasmid provides a powerful tool for genetic and
biochemical analysis of RNAP. The resulting enzymes are substantially
pure after two steps, primarily because chitin affinity chromatography
is much more selective than the
hexahistidine-Ni2+-nitrilotriacetic acid-agarose method
that has been widely used to date (44), and has the advantage of being
assembled by the normal pathway in vivo. We anticipate that
the co-overexpression plasmid will also prove useful for selection of
RNAP suppressor mutants of inviable amino acid substitutions in the
enzyme. Finally, because RNAP is pure enough for in vitro
transcription assays after just the single chitin column step, the
method should facilitate rapid screening of sets of closely related
alterations to RNAP.
In using the co-overexpression method for mutant RNAP assembly and
purification, we encountered a few problems that require care to avoid.
First, the chitin matrix apparently can break down when batch binding
and elution are used, which prevents retention of the tagged enzyme on
the solid chitin matrix. We used slow passage of lysates through a
column of chitin matrix to avoid this problem. Second, the efficiency
of DTT-mediated cleavage of the Sce VMA intein that connects
' to the CBP was usually less than 100%, and both the binding
capacity and the cleavage efficiency appeared to vary among lots of
chitin matrix. It is advisable to test new batches of matrix before
committing valuable samples; some loss of material due to inefficient
cleavage appears unavoidable. Third, we sometimes observed loss of
expression of one or more of the RNAP subunits when the co-expression
plasmid was maintained in recA+, T7
RNAP-expressing strains, apparently due to plasmid rearrangements, mutations, or recombination with the chromosomal copies of
rpoA, rpoB, or rpoC. The last problem
was substantially eliminated by maintaining the plasmids in
recA strains such as DH5
except during expression.
Finally, we emphasize that not all RNAP mutants can be successfully
recovered by the co-overexpression approach. For instance, a deletion
slightly larger than
'
SI3 (
932-1137 versus
943-1130 in
'
SI3) failed to assemble and yielded only
insoluble
'
(932-1137) aggregates in vivo.
Role of 'SI3 in RNAP Function--
Although both SI1 and SI2 in
exhibited some effects on the biochemical properties of RNAP (and
were essential for full viability in vivo), the most
dramatic effects on RNAP properties were observed for
'SI3.
'SI3
profoundly affected initiation and pausing by E. coli RNAP,
in both cases accounting for many but not all of its differences from
B. subtilis RNAP. We will consider the effects on initiation
and pausing separately, although the underlying changes in RNAP's
properties could be related.
'SI3 stabilized open complexes formed at the T7 A1 promoter against
disruption by heparin. This stabilization could reflect either direct
contact with the downstream DNA, as previously proposed (16), or it
could reflect indirect stabilization of the open complex if folding of
'SI3 is coupled to structural transitions in RNAP that occur upon
open complex formation. Although direct DNA interaction is an
attractive hypothesis, no cross-linking studies to date unambiguously
document interaction of
'SI3 with the downstream DNA in open
complexes. In the recently published crystal structure of T. thermophilis RNAP, the loop in
'G to which
'SI3 connects is
positioned near the downstream DNA channel (11), also favoring its
possible interaction with downstream DNA. However, this structure is of
an enzyme that lacks
'SI3 and was obtained in the absence of DNA.
The
'G loop is poorly ordered in both the yeast RNAPII and T. aquaticus RNAP crystal structures and was found in different
conformations in the ordered portions of the two structures, suggesting
that it is capable of significant motion. Further,
'SI3 itself is
not visible in a recent electron microscopy crystal structure of
E. coli RNAP, suggesting that it is even more mobile than
the
'G loop (10). Thus,
'SI3 folding could be coupled either
directly or indirectly to interactions of RNAP with downstream DNA.
Many DNA-binding proteins undergo folding transitions in protein
segments distant from the actual DNA binding surface (45). These
indirect but coupled protein folding events are thought to contribute
significantly to the avidity of protein-DNA interactions. Thus,
stabilization of open complexes by
'SI3 could arise not via its
direct interaction with the downstream DNA but via an indirect coupling
of its folding to open complex formation. Further study will be
required to distinguish these possibilities.
Both the direct and indirect explanation of 'SI3 function also could
apply to its effect on transcriptional pausing. Both formation of
paused transcription elongation complexes and their slow
escape are enhanced by an interaction of downstream DNA with RNAP
(46-49). Thus,
'SI3 could strengthen the pause-enhancing downstream
DNA interaction either directly or by the indirect mechanism described
above. Interestingly, the effects on pausing of deleting
'SI3 are
quite similar to the effects of deleting the
' jaw domain of
E. coli RNAP, for which a variety of data suggest a direct
DNA interaction (49).
In the case of pausing, however, a third explanation is possible.
'G, in which
'SI3 is inserted, appears to cross-link to the RNA
3' nt in the his paused transcription
complex3 as well as to the
RNA 3' nt in arrested complexes (50) and in certain artificial
transcription complexes that may mimic paused transcription complexes
(51). Further, amino acid substitutions in
'G, near the site of
'SI3 insertion, strongly affect chain elongation and transcriptional
pausing and termination (40, 51). If movements of the
'G loop are
involved in transcriptional pausing, it could readily explain why
deletion of
'SI3 has such a strong effect on pausing. This also
might explain why a partial deletion in
'SI3 (
1091-1130) as well
as monoclonal antibody binding to
'1091-1130 in wild type RNAP
greatly increase pausing (14), whereas complete deletion of
'SI3
(
943-1130) decreases pausing (Figs. 4 and 5). By altering the
structure of
'SI3, both
'
(1091-1130) and antibody binding
could interfere with movements of the
'G loop necessary for rapid
nucleotide addition, whereas removal of
'SI3 could favor such movements.
Roles of SI1 and
SI2 in Abortive Initiation--
SI1 and
SI2 both exhibited effects on initiation complexes, either in open
complex longevity, abortive initiation, or both. Interestingly, both
SIs increased abortive initiation significantly, although they
decreased open complex longevity modestly (
SI1) or had no effect on
it (
SI2). This suggests that RNAP must be exceptionally sensitive to
changes in its structure during abortive initiation. This could be
related to the sequential rearrangements of contacts between core RNAP
and
that are thought to occur during the initial stages of RNA
synthesis (52). Perhaps
SI1 and
SI2 somehow facilitate the
core-
rearrangement during initial transcript synthesis.
Alternatively, they could indirectly affect the conformation of the
main channel of RNAP such that their removal increases the rate of
abortive transcript release.
Sequence Insertions Are Ubiquitous in Bacterial
RNAPs--
Finally, we note that sequence insertions are not limited
to those found in the RNAPs from enteric and thermophilic bacteria. A
search of sequences now available for and
' subunits in bacteria revealed that sequence insertions are ubiquitous and that the positions
of these insertions are not restricted to those observed in the enteric
and thermophilic bacteria (Figs. 1 and
6). Rather, the sequence insertions
differ in size, sequence, and location but are predicted to be
surface-exposed (Fig. 6). Such a distribution of sequence insertions is
consistent with the idea that they confer species-specific properties
on core RNAP, either by modulating its enzymatic activity directly or
through interactions with transcription factors. The relatively low
degree of divergence among SIs in closely related proteobacterial
species (see Introduction) also lends support to this idea.
|
This pattern of surface-exposed sequence insertions is reminiscent of
the small subunits of eukaryotic RNAPs, which also are surface-exposed
(at least in RNAPII); vary in composition to some extent among RNAPI,
RNAPII, and RNAPIII; and can affect the biochemical properties of the
enzyme yet are dispensable for core function. The RNAPII subunits 4 and
7, for instance, are readily dissociated from RNAP and affect its
biochemical properties but are dispensable for nucleotide addition (53,
54); deletion of RNAPII subunit 9, like deletions in 'SI3, affects
responses of the RNAPs to their respective RNA cleavage factors, GreB
(14) and TFIIS (55). Perhaps the bacterial sequence insertions are like
these small eukaryotic RNAP subunits, conferring distinctive properties
on the RNAPs of different bacterial species much as the small RNAP subunits may confer distinctive properties on RNAPI, RNAPII, and RNAPIII.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Seth Darst for help with molecular modeling and Josefine Ederth, Kati Geszvain, Rachel Mooney, Ruth Saecker, and Kesha Toulokhonov for helpful comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM38660 and Department of Agriculture Grant WIS04022 (to R. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Institutes of Health Grant GM 28575 (to Richard R. Burgess, Department of Oncology, University of Wisconsin, Madison, WI).
** To whom correspondence should be addressed. Fax: 608-262-9865; E-mail: landick@bact.wisc.edu.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M211214200
2 Sequence insertions in E. coli RNAP previously have been designated DR, to signify dispensable regions (see Ref. 7 and references therein). We prefer the less restrictive abbreviation SI to signify lineage-specific sequence insertions, because E. coli cells containing SI deletions are inviable in at least some conditions and exhibit altered biochemical properties, suggesting that these features of RNAPs play important functional roles. Further, the SI nomenclature is applicable to other bacterial RNAPs for which functional tests have not been performed.
3 K. Toulokhonov and R. Landick, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
RNAP, RNA
polymerase;
SI, lineage-specific sequence insertion;
ORF, open reading
frame;
CBP, chitin-binding protein;
nt, nucleotide(s);
aa, amino acid(s);
IPTG, isopropyl-1-thio--D-galactopyranoside;
DTT, dithiothreitol.
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