 |
INTRODUCTION |
RNA polymerase (RNAP)1
from bacteria consists of a multisubunit enzyme complex that is the
main target for the regulation of gene expression. The catalytic core
of RNAP is composed of the subunits
,
', and
and is
sufficient to catalyze the polymerization of NTPs into RNA chains. In
addition, a vast array of other proteins makes contacts with RNAP at
one or more steps of the transcription cycle and modifies its
activities in various ways (1). Such ancillary polypeptides modify RNAP
during the promoter binding, initiation, elongation, and termination
steps to effect changes in gene expression. The affinity of the various
transcription factors for RNAP varies greatly during the transcription
cycle; for example,
factors have a high affinity for RNAP during
the pre-initiation steps but very low affinity during elongation, whereas for NusA the opposite occurs (2).
In the Gram-positive bacterium Bacillus subtilis, purified
preparations of RNAP are usually found associated with the 21.4-kDa
factor (3, 4).
was found to be essential, together with a
phage-encoded
factor, to reconstitute selective transcription of
the phage SP01 genome in vitro (3). Further biochemical characterization of
activity has revealed complex effects on RNAP
activity. For example,
reduces the extent of contact between RNAP
and promoter DNA, as detected by DNA footprinting assays, and
antagonizes the formation of the open complex (5-8). These results
suggest that RNAP binds to the promoter as a E
complex. However,
in solution
and
bind to RNAP with negative cooperativity (9).
In addition,
can either inhibit or stimulate in vitro transcription, depending on reaction conditions, and its stimulatory effects are clearly DNA template-dependent (7, 10-13).
has a very unusual tertiary structure with an amino-terminal half
that is folded as a
/
type of structure and a carboxyl-terminal half that is unstructured in solution, as observed by circular dichroism analysis (14). The carboxyl-terminal region is highly acidic,
consisting mostly of aspartate (37%), glutamate (34%), and
hydrophobic residues (25%) and is essential for displacing RNA bound
to RNA polymerase (14). This RNA displacement activity may explain our
previous observation that
enhances RNAP recycling in multiple cycle
transcription reactions, thus stimulating overall transcription (13).
These structural and functional studies suggest that
acts by
mimicking single-stranded RNA and points to a role of
in the
termination or recycling steps of transcription (14).
Despite the clear effects of
on in vitro transcription,
initial attempts to demonstrate a role for
in vivo were
unsuccessful.
mutant strains sporulate, lack auxotrophies, grow up
to 49 °C, and plate phage SP01 normally (4). In this study, we
describe the expression of the rpoE locus, the phenotype of
an rpoE deletion mutant, and preliminary biochemical
analysis of the interactions between
and RNAP.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Restriction enzymes and T4 polynucleotide kinase
were purchased from New England Biolabs, Inc.; avian myeloblastosis
virus reverse transcriptase was from Promega Corp.; growth media were from Difco; and [
-32P]dATP and
[35S]methionine were from NEN Life Science Products.
Growth Conditions--
Strains were grown with vigorous shaking
at 37 °C in Luria broth (LB) (15) or in minimal medium (16), which
is composed of 40 mM MOPS buffer, pH 7.4, 2% glucose
(w/v), 2 mM potassium sulfate, pH 7.0, 10 µg/ml
tryptophan, and 1× Bacillus salts (0.2 g of magnesium
sulfate, 2 g of ammonium sulfate, 1 g of sodium citrate, and
1 g of potassium glutamate/liter). Antibiotic concentrations used
were, for Escherichia coli, ampicillin at 100 µg/ml; for B. subtilis, erythromycin at 2 µg/ml; neomycin, 10 µg/ml; macrolides/lincomycin/streptogramin B with 1 µg/ml
erythromycin and 25 µg/ml lincomycin.
Bacterial Strains--
B. subtilis strains used were
CU1065 (W168 trpC2 attSP
), HB6002 (CU1065
rpoE::lacZ, generated using the pTKlac vector),
HB6005 (CU1065 PrpoE::cat-lacZ at the
SP
locus, generated using the pJPM122 vector (17)), HB6010 (CU1065
rpoE::cm, generated by transformation of CU1065 with RP17 DNA (14)), HB6012 (HB6002 abrB::neo), and HB6013 (HB6002
sinR::kan). The rpoE mutant
used in this study is a deletion/insertion containing a chloramphenicol resistance cassette replacing the segment of the rpoE gene
between codons 6 and 142 (4). E. coli strains used were
DH5
(supE44
lacU169
80
lacZ
M15 hsdR17 recA1
endA1 gyrA96 thi-1 relA1)
and, for protein overexpression, BL21/DE3 (BL21 with
DE3 (18)).
Generation of rpoE-lacZ Fusions--
Plasmids used for the
generation of lacZ fusions to the rpoE promoter
region were pTKlac (19) and pJPM122 (17). The cloned rpoE
gene was obtained originally from plasmid pCB
11 (4), a pBR322
derivative containing rpoE on a ~2-kilobase
HindIII fragment. To generate a transcriptional fusion to
lacZ at the rpoE locus, the
rpoE-containing HindIII fragment from pCB
11
was cloned into pBSKII+ (Stratagene) such that the
rpoE gene was oriented toward the unique KpnI
site. The resulting plasmid, pFL22, was digested with EcoRI
and BglII, and the promoter-containing fragment was ligated to pTKlac and digested with EcoRI and BamHI to
generate pFL23. Integration of pFL23 into the rpoE mutant
strain RP3 (4) generates strain HB6001 (rpoE-lacZ), whereas
integration into the wild type (CU1065) generates
HB6002(rpoE-lacZ). For generating the lacZ transcriptional fusion at the SP
locus, a HindIII to
SspI fragment of pFL20 (14) was cloned into pBSKII+ after
digestion with HindIII and SmaI to generate
pFL21. The promoter-containing fragment was excised by digestion with
HindIII and BamHI and cloned into pJPM122 after
digestion with the same enzymes to generate pFL60. pFL60 was first
integrated into B. subtilis ZB307A (20), and a transducing lysate was used to transfer of the
PrpoE-cat-lacZ operon fusion into wild type
(CU1065) and rpoE mutant (HB6010) backgrounds to generate
strains HB6005 and HB6006, respectively.
Overexpression of
in B. subtilis--
Overexpression of
in B. subtilis was achieved using the
Pspac-containing plasmid, pDG148 (21). The
rpoE gene was removed from plasmid pFL32 (14) as an
EcoRI to EcoRV fragment and cloned into
pBSKII+ after digestion with EcoRI and
SmaI to generate pFL24. Then the rpoE gene was
removed from pFL24 as an XbaI fragment and cloned into
pDG148 after digestion with XbaI to generate pFL25.
Primer Extension Analysis--
RNA was purified using the RNeasy
Total RNA kit purification system of Qiagen Inc. RNA from in
vitro reactions was produced by mixing a polymerase chain reaction
(PCR) fragment containing the rpoE promoter with 1 pmol of
Bacillus RNA polymerase core enzyme supplemented with 10 pmol of purified
A, 1× transcription buffer (40 mM Tris acetate, pH 8.0, 50 mM ammonium acetate, 20 mM potassium acetate, 4 mM
magnesium acetate, 0.1 mM dithiothreitol, and 0.04 mg/ml
bovine serum albumin), and 0.1 mM NTPs, and incubating the
mixture at 37 °C for 15 min. The "run-off" RNA was then purified
using the Qiagen kit and precipitated with ethanol. A primer
(5'-TCCTTTAGCTCTTCCTG-3'), centered at around 40 base pairs downstream
of the translation start site, was labeled with
[
-32P]ATP and polynucleotide kinase (New England
Biolabs, Inc.) following standard procedures (15). The labeled primer
was then hybridized to the RNA in hybridization buffer (60 mM NaCl, 50 mM Tris-Cl, pH 8.0, and 10 mM dithiothreitol) by heating to 90 °C for 1 min and
then cooling to 25 °C over 2 h. Next, nucleic acids were
precipitated with ethanol in the presence of 0.3 M sodium
acetate, pH 5.2, redissolved in extension buffer (50 mM
Tris-HCl, pH 8.3, 40 mM potassium chloride, 7 mM magnesium chloride, 1 mM dithiothreitol, and
0.1 mg/ml bovine serum albumin) supplemented with 10 mM
dNTPs and 10 units of avian myeloblastosis virus reverse transcriptase, and incubated at 37 °C for 20 min. The resulting labeled DNA was then visualized by electrophoresis followed by autoradiography.
Deletion of the rpoE 5'-untranslated Region--
To delete the
5'-untranslated region preceding the rpoE gene, the promoter
region was amplified using the PCR and pFL24 as a template with
primers NY1
(5'-GGCCCGAAGCTTTAACGGAAAACATCTCTCAGTCGG-3') and NY2
(5'-CGGGAATTCCTTATACAAACCATACCTCTC-3'), and the resulting product was digested with HindIII and EcoRI
(sites underlined). The 5'-end of the rpoE gene,
together with the corresponding ribosome binding site, was amplified
using primers NY3
(5'-GGCGAATTCTAGAAAGGGAGTGTCCGACCTTGGG-3') and #107
(5'-GCGGATCCTACTTGACTGTCGGCTGAG-3') and digested with EcoRI and BamHI. The two PCR products were cloned
in a three-way ligation into pJPM122 digested with
HindIII and BamHI to generate pNY50.
Transformation of pNY50 (linearized with ScaI) into ZB307A generates HB6040. Transducing lysates, prepared from HB6040, were used
to move the SP
-borne PrpoE
-cat-lacZ operon
fusions into CU1065 (generating HB6041) and the rpoE mutant
strain HB6010 (generating HB6042).
-galactosidase Assays--
To assay gene expression, rich
medium (2xYT) or minimal medium was inoculated from an overnight
culture by 1:100 dilution, samples were taken, and
-galactosidase
was assayed by the procedure of Miller (22).
Western Blots--
Immunoblots were carried out using rabbit
anti-
antibodies (14), goat horseradish peroxidase-coupled
anti-rabbit secondary antibodies (Bio-Rad), and an enhanced
chemiluminiscence detection system (NEN Life Science Products)
following the instructions of the suppliers. Pure
was obtained as
described (14), and quantitation of its concentration was carried out
using the Bradford reagent (Bio-Rad). Extracts of B. subtilis CU1065 cells were prepared by sonication of cells after a
treatment with lysozyme and centrifugation at 12,000 rpm for 10 min.
Spores of CU1065 and HB6010 were prepared by extensive washing, and
extracts were prepared as described (23).
Construction and Purification of PKA
Derivatives--
Derivatives of
and
A were
constructed by the addition of a protein kinase A recognition motif to
the amino terminus of the protein (24). We used the PCR to place 11 additional codons at the 5'-end of the corresponding genes.
Oligonucleotides used for PCR amplification of the
gene and
addition of the PKA sequence were a "PKA primer"
(5'-AAAAGCTAGCCTGCGTCGTGCGTCCCTGGGTGATCAGGGTATCAAACAATATTCA-3') and a 3'-rpoE primer
(5'-CGCGGATCCCGACTATGAAAGTCAAGATCG-3'). The PKA primer
encodes a PKA recognition site (ASLRRASLGDE) between a
5'-NheI site (singly underlined) and a BclI site
(doubly underlined) used in later constructions. The PCR product was
cloned first into pET11c (Novagen) as an NheI to
BamHI fragment to generate pFL70. To generate
NPKA and
CPKA, we cloned the
rpoE gene from the pFL70 product as an XbaI to
BamHI fragment into pBKSII+ (digested with
XbaI and BamHI) to generate pFL80. For
NPKA we digested pFL80 at two BglII sites
internal to the rpoE gene, filled the recessed termini using
the Klenow enzyme and all four dNTPs, and ligated the resulting blunt
termini with T4 ligase, thus creating a new BspDI site and
an in-frame stop codon in plasmid pFL82 as described previously for
unmodified rpoE (14). To create
CPKA, we
digested pFL80 with BclI, partially digested with
BglII, and ligated. A plasmid was identified with a deletion
of the amino-terminal 109 amino acids of
and was designated pFL85.
For protein overproduction, each modified rpoE gene was
removed as an XbaI to BamHI fragment and cloned
into pET11c to generate plasmids pFL72 (
NPKA) and pFL75
(
CPKA). The modified rpoE genes were verified
by DNA sequencing.
For the construction of
APKA the following
oligonucleotides were used as PCR primers in a reaction containing the
cloned sigA gene as template:
5'-GGGCTCTAGACTGCCTCGTCGCTCCCTGGCTGATAAACAAACCCACGAC-3' and 5'-CGCGGATCCTTAATCGTCCAGGAAGCTACG-3'. The resulting PCR
product was digested with XbaI and BamHI (sites
underlined) and ligated into pET11c digested with NheI and
BamHI to generate pFL90. The sequence of the modified
sigA gene was verified by automated DNA sequencing.
Purification of
PKA and
APKA
was as described for the unmodified proteins (14, 25). Briefly,
NPKA was purified from E. coli BL21/DE3 cells
by passage of an extract of
isopropyl-1-thio-
-D-galactopyranoside-induced cells
through an S-Sepharose chromatography column previously equilibrated
with buffer A (50 mM HEPES, pH 8.2, 2 mM EDTA,
and 5% glycerol in water). After washing and elution of
NPKA with buffer A supplemented with 200 mM
NaCl, the protein solution was precipitated with ammonium sulfate (60%
saturation) and centrifuged. Further purification of
NPKA was carried out by the passage of the protein
through fast protein liquid chromatography Superdex 70 (size exclusion
chromatography) and Mono-S (ion exchange chromatography) columns. PKA
derivatives were labeled with [
-32P]ATP and protein
kinase A (New England Biolabs) as described (24).
 |
RESULTS |
The Structure of the rpoE Promoter Region--
The rpoE
promoter region contains a predicted
A-type
promoter element located ~90 base pairs upstream of the translational start site (Fig. 1A). Primer
extension analysis (Fig. 1B) with RNA purified from
vegetatively growing cells and from in vitro transcription
assays using B. subtilis RNA polymerase containing
A shows that transcription starts with approximately
equal frequency at either of two purine residues located 84 and 86 base
pairs from the translation start site. The putative
10 region of the promoter conforms to a
A consensus promoter (26), but
there is weak if any similarity in the
35 region.

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 1.
Identification of the promoter for
rpoE. The sequence of the rpoE
promoter region (A) is illustrated from 58 to +91 relative
to the first transcription start site (+1). The two main transcription
start sites are marked by arrows. The 10 region of the
promoter and the putative ribosomal binding site (r.b.s.)
are capitalized. The initial transcribed region can assume
at least two possible secondary structures, and the corresponding
inverted-repeat sequences are indicated (><). The extent
of the deletion engineered in strains HB6041 and HB6042 is
overlined and marked with a . Note that the
gene begins with a TTG start codon. B shows primer
extension analysis of the rpoE promoter using RNA template
purified from cells (lane 1), RNA generated in
vitro using B. subtilis core RNAP supplemented with
purified A (lane 2), or RNA generated
in vitro with the addition of purified to the
transcription reaction (lane 3).
|
|
Expression of rpoE and Abundance of
in the Cell--
The level
of expression of rpoE was monitored by
-galactosidase
assays of cells containing rpoE-lacZ transcriptional
fusions. When measured at the rpoE locus (strain HB6002),
expression reaches a maximum of 60-80 Miller units at the transition
between logarithmic and stationary phase (Fig.
2). Other strains in which the fusion was
placed at the SP
locus, either in wild type or in an rpoE mutant, produced essentially identical results (data not shown).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Expression of the rpoE
gene. -galactosidase activity was measured in a strain
carrying a rpoE::lacZ fusion, HB6002
( , right axis) as a function of growth phase as measured
by optical density ( , left axis). The x axis
indicates time after inoculation (hours). A reproducible increase in
expression is observed at the end of logarithmic growth.
|
|
Folding of the untranslated RNA molecule with the Mulfold program (27)
shows that this region contains several inverted repeats predicted to
form stable stem loops (estimated
G of
23.3 and
22.7 kcal/mol at
25 °C and 1 M NaCl) (Fig. 1A). To investigate the possible regulatory function of these sequences, a deletion mutation was engineered and placed in wild type (HB6041) or
rpoE mutant (HB6042) cells.
-galactosidase analysis of
these two strains showed that rpoE expression is increased
2-fold in the absence of these sequences. However, as noted for the
wild-type leader region, the presence or absence of
had no
detectable effect on expression. In vitro transcription from
the rpoE promoter showed that small RNA fragments about 50 nucleotides long are produced when these sequences are present,
indicating that these stem loops might function as transcriptional
terminators. As with the in vivo result, the presence or
absence of
in the transcription reaction did not make a difference
(data not shown). Thus, expression of rpoE is unaffected by
, and we find no apparent autoregulation of this locus.
Quantitative immunoblots were performed to estimate the abundance of
protein. The antiserum used for these studies is highly specific
and reacts with a single 21-kDa band present in wild-type cells but
absent from rpoE mutant strains (14) (Fig.
3B, lane 1). When 10 µg of
cell lysate is analyzed, the amount of
detected remains constant
during growth at between 10 and 50 ng (Fig. 3A, lanes
1-3) as judged by comparison with the pure protein. We therefore estimate that
represents 0.3 ± 0.1% by weight of soluble
cell protein. Because RNA polymerase (Mr = 337,000) represents 1% by weight of soluble cell protein (28), we
conclude that
is an abundant protein (~104
molecules/cell) present in an approximately 5:1 molar excess relative
to RNAP. Purified spores also contain similar levels of
(Fig.
3B, lane 2).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 3.
Estimation of the abundance of
in vivo. Immunoblot analysis,
using anti- antibodies, is presented for extracts from vegetative
cells (A) and spores (B). A,
lanes 1-3, 10 µg of soluble cell protein from B. subtilis CU1065 grown in LB medium supplemented with glucose and
collected at mid-logarithmic phase, A600 0.3 (lane 1); transition state, A600 0.7 (lane 2); and early stationary phase,
A600 0.9 (lane 3). Purified was
loaded in lane 4 (10 ng), lane 5 (50 ng), and
lane 6 (100 ng) as standards. In B, 10 µg of
soluble protein extracted from spores of the rpoE mutant
HB6010 (lane 1) or the wild-type CU1065 (lane 2)
were loaded.
|
|
The Phenotype of the rpoE Mutant--
The fact that the
rpoE gene seems to be up-regulated in the transition state
between the logarithmic and stationary phase (Fig. 2) might be
indicative of a role of
in stationary phase phenomena. To
investigate the consequences of an rpoE disruption on cell
growth, we chose a mutation (RP17) in which most of the rpoE
coding sequence is deleted (4). We find that rpoE mutants generate a striking colony morphology when incubated for extended times
(3-4 days) on Luria agar at room temperature; colonies of HB6010 show
a rough edge with a fractal geometry (Fig.
4B) in contrast to the
isogenic parental strain, which has smooth edged colonies. In addition,
after dilution of 24-h-old cells into fresh medium (1:100), the cells
of HB6010 are markedly more elongated and tend to occur in aggregates,
as seen under the light microscope (Fig. 4D), and this
phenotype persists for several generations. HB6010 also shows a
reproducible 30-min delay in entrance into the logarithmic phase (Fig.
5), as compared with CU1065, in either minimal medium or LB growth medium.

View larger version (93K):
[in this window]
[in a new window]
|
Fig. 4.
An rpoE mutant has an
altered morphology. Growth pattern on agar plates of strains
CU1065 (A) and HB6010 (B). Cells were diluted
from an overnight culture, plated on Luria agar, and incubated at
37 °C for 12 h and at room temperature for a further 48 h.
C and D, cells were grown overnight at 37 °C
with vigorous shaking, diluted in fresh medium, and grown to an
A600 of 0.2 (early logarithmic phase). In
HB6010, a rpoE deletion mutant, cells were aggregated
(clumped) and elongated with respect to wild type, features that
gradually disappeared as the cultures reached an
A600 of 0.5 and above.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
An rpoE mutant has a
lengthened lag phase. Growth curves of strains CU1065 ( ) and
HB6010 ( ) when diluted 100:1 in fresh liquid LB medium after a
24 h incubation at 37 °C with vigorous shaking. Viable cell
counts (colony forming units) demonstrated that viability is identical
for both strains even after prolonged incubation. The average
difference of time of entrance into logarithmic growth between CU1065
and HB6010 is 30 min.
|
|
In contrast with these results, most other attempts to define a
phenotype for the rpoE mutant strain have not been
successful. HB6010, a rpoE deletion mutant, sporulates with
the same efficiency as CU1065, a wild-type strain, and the purified
spores are equally sensitive to UV irradiation (data not shown). Growth
rate in Luria broth, sporulation medium, or minimal medium is also
indistiguishable between the two strains, as originally found by Lampe
et al. (4). We did not detect any differences in motility or
in resistance to H2O2 between CU1065 and HB6010
(data not shown). Double mutations of rpoE with other known
regulators of transition state phenomena, such as abrB or
sinR, did not result in phenotypes distinguishable from the
phenotypes because of these mutations alone (data not shown). Finally,
induction of
from the
isopropyl-1-thio-
-D-galactopyranoside inducible
Pspac promoter (in pFL25) led to the
overproduction of
protein as observed by immunoblot analysis but
did not produce any appreciable changes in the growth rate or viability
nor did it have any major effects on protein composition of the cell, as judged by a [35S]methionine pulse-chase experiment
followed by SDS-polyacrylamide gel electrophoresis (data not shown).
Affinity of
for RNAP Core--
For use in biochemical studies,
we placed a 9-amino acid recognition site for protein kinase A at the
amino terminus of
to generate
PKA. Overexpression
and purification of
PKA resulted in a protein that
displaced nucleic acids as efficiently as wild-type
(14).
We used 4% native polyacrylamide gel electrophoresis to study the
interaction of
PKA with RNAP. As expected for a small
protein with a
49 net charge,
PKA migrated very fast,
but its mobility was greatly reduced in the presence of increasing
amounts of core RNAP (Fig. 6).
Interestingly, two very closely spaced bands are observed suggesting
that two distinct forms of RNAP-
complex are present. Similar
results are seen with E. coli RNAP (data not shown).

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 6.
Electrophoretic mobility shift assay of
-PKA binding to RNAP. protein (10 fmol)
was labeled with 32P and mixed in a reaction with
increasing concentrations of core RNAP from B. subtilis.
Lane 1, no RNAP added. Lanes 2-6 contain 0.35, 0.7, 1.4, 2.8, and 5.8 pmol of RNAP, respectively, added in a 5-µl
reaction. Buffer conditions are as described by Altmann et
al. (38). All reactions were carried out at room temperature.
Separation of free from complexed followed in a 4% native
polyacrylamide gel run in Tris-borate EDTA buffer (15).
|
|
Under these conditions 0.28 µM core enzyme binds less
than 50% of the labeled
, whereas 0.54 µM binds
nearly all of the labeled
. Because
is present at very low
levels (2 nM) in these reactions, we can estimate the
apparent dissociation constant as the concentration of RNAP required
for half-maximal binding (assuming all RNAP can bind
). By
interpolation, this is ~0.4 µM, corresponding to an association constant of 2.5 × 106
M
1. The presence of a single complex upon the
addition of RNAP is consistent with a stoichiometry of 1:1 as
previously observed (9, 14).
Effects of DNA and
on the Interaction of
with RNAP--
We
used labeled
PKA and
PKA to investigate
the interactions of
and
with RNAP in the presence and absence
of the strong trnS promoter (Fig.
7). As before, the addition of core RNAP
(lane 2) leads to the appearance of a slower mobility
species (E
). To see whether we could detect formation of a E
complex, we added an excess of unlabeled
A. However,
under these conditions, there was no "supershift" detected, suggesting that
A was unable to bind the E
complex
(lane 4). However,
is displaced from the core by the
presence of DNA (lanes 3 and 5). In a parallel series of experiments using radiolabeled
APKA, we observed the formation of a slow
mobility complex corresponding to promoter-bound E
A
(lanes 7 and 9) (data not shown), and this
complex is stable to the addition of excess cold
. We again failed
to detect a
-dependent supershift, suggesting that,
under the conditions tested, binding favors complexes of E
or
E
·DNA but not E
·DNA or E
·DNA.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 7.
Electrophoretic mobility shift assay of
PKA and
APKA interactions with
RNAP. Under conditions similar to those described in Fig. 6, we
used native 4% polyacrylamide gel electrophoresis to separate free
from bound 32P-labeled APKA and
PKA in complexes with RNAP in the presence and absence
of promoter DNA. On the left side of the panel, 10 fmol of
labeled PKA was mixed with RNAP dialysis buffer
(lane 1) or 0.1 pmol of B. subtilis core RNAP
(lanes 2-5) in 5-µl reactions. In lanes
3-5, 1 pmol of a 165-base pair DNA fragment containing
the trnS promoter of B. subtilis (8),
APKA (1 pmol, unlabeled), or both was added
as indicated. Lanes 6 and 7 contain 0.1 pmol of
32P-labeled A. RNAP (0.1 pmol), DNA (1 pmol), and unlabeled PKA (0.5 pmol) were added as
indicated.
|
|
 |
DISCUSSION |
Previous analyses indicated that
, a stoichiometric component
of RNAP purified from B. subtilis, is dispensable for
growth. Indeed, a rpoE mutant had no apparent phenotype (4).
Nevertheless,
has large effects on the biochemical properties of
RNAP (3, 5-8, 10-14, 29). In this paper, we confirm and extend these previous observations of the rpoE mutant phenotype,
quantitate the levels of
in growing cells, and characterize the
promoter region driving
expression. Finally, we demonstrate
biochemically that
interacts with RNAP with an affinity comparable
to other dissociable subunits.
Phenotype of the rpoE Mutant Strain--
Our analyses have
revealed that an rpoE deletion mutant is unaltered in growth
rate or viability under numerous conditions. However, we reproducibly
detect an extended lag phase in the rpoE mutant strain,
suggesting that
may play a role during growth phase transitions.
This notion is also consistent with the observation that
is
maximally expressed during the transition from growth to stationary
phase. In addition, rpoE mutant cells retain an elongated
morphology and tend to clump during early logarithmic phase growth.
When grown on solid medium, the rpoE mutant has an enhanced
tendency to produce rough edged colonies. The factors affecting colony
morphology are complex (30, 31) and not well understood, so this
phenotype is not very informative.
The relatively modest phenotype associated with an rpoE
mutation is surprising for a subunit of RNAP. Another protein may be
functionally redundant with
, but attempts to identify such a
protein by transposition mutagenesis were not successful (data not
shown). Moreover, the availability of the complete genome sequence of
B. subtilis does not reveal any paralogs (32). Nevertheless,
is conserved in several other Gram-positive organisms including Mycoplasma genitalium, Mycoplasma pneumoniae,
Enterococcus faecalis, and Streptococcus
pyrogenes. These homologs contain a statistically significant
similarity to the amino-terminal domain of
and are typically
associated with an acidic carboxyl-terminal domain. Previous work has
revealed functionally equivalent proteins in other Bacilli
(33), and a similarly sized protein was found to co-purify with RNAP
from Staphylococcus aureus (34). Indeed, mutations in an
S. aureus gene encoding a putative
homolog lead to a
stationary phase survival defect (35). Thus,
seems to be a
bona fide subunit of RNAP found in many different
Gram-positive bacteria that may be important for long term survival or
during growth phase transitions.
Interactions of
with RNAP--
is abundant in the cell
during all stages of growth, at concentrations of approximately
104 copies/cell. This concentration would be sufficient to
saturate the estimated amounts of RNAP in the cell. It also seems to be an abundant protein in spores, although the rpoE mutant
sporulates and germinates with equal efficiencies as the wild type, and
its spores are equally resistant to UV light. Induced expression of
from a plasmid did not alter patterns of gene expression in B. subtilis cells. Expression of rpoE is driven from a
single
A-dependent promoter and is
essentially constitutive under the conditions tested. We found no
evidence for autoregulation.
Previous analyses had established that
, by virtue of its
polyanionic carboxyl-terminal region, competes with nucleic acids for
binding to RNAP (14). However, it was also observed that a
rpoE mutant strain containing a truncated gene produced a
stable protein corresponding to the amino-terminal domain of
and
that this domain still co-purified with RNAP. Thus, determinants for core binding are likely to be present in both the amino- and
carboxyl-terminal regions (14).
co-purifies with RNAP through
several chromatographic steps and during late stages of purification
tends to partition with fractions of RNAP containing alternative
factors and not
A (36). In an in vitro
competition assay, the presence of
biases the binding of the
alternative
factor gp28 (phage SP82) to RNAP over
A
(9). Thus, a role for
in
switching can be envisaged. A role for
in the events leading to the recycling of RNAP after transcription
termination has also been documented;
has the unique ability of
displacing RNA from binary complexes with RNAP, and this effect
requires the presence of the carboxyl-terminal half of
,
C, which
mimics RNA (14). Despite the abundant evidence indicating effects of
at the initiation complex (5, 6, 8, 11-13, 29), we were unable,
using an electrophoretic mobility shift assay, to document the
formation of the presumed E
complex in either the presence or
absence of promoter DNA. This is consistent, on the other hand, with
previous studies documenting negative cooperativity between
and
(9) and with the fact that
mimics RNA and RNA displaces
from
RNAP (37).
Concluding Remarks--
The physiological role of
remains a
mystery, although biochemical evidence suggests that this protein
likely participates in both the initiation and recycling phases of
transcription. As proposed in early studies, it is not yet excluded
that
may function in
factor switching, but such a role is
clearly not essential. B. subtilis is now known to harbor no
fewer than 17
factors including several whose functions are only
just beginning to be deciphered. It will be interesting to determine if
rpoE mutations are epistatic with any of these newly
described regulators.