From the Rockefeller University, New York, New York 10021 and
the
Waksman Institute, State University of New Jersey,
Rutgers, New Jersey 08855
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ABSTRACT |
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During the development of purification procedures
for Escherichia coli RNA polymerase (RNAP), we noticed the
consistent co-purification of a 110-kDa polypeptide. Here, we report
the identification of the 110-kDa protein as the product of the
hepA gene, a member of the SNF2 family of putative
helicases. We have cloned the hepA gene and overexpressed
and purified the HepA protein. We show in vitro that RNAP
preparations have an ATPase activity only in the presence of HepA and
that HepA binds core RNAP competitively with the promoter specificity
70 subunit with a 1:1 stoichiometry and a dissociation
constant (Kd) of 75 nM. An E. coli strain with a disruption in the hepA gene shows
sensitivity to ultraviolet light.
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INTRODUCTION |
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DNA-dependent RNA polymerase (RNAP)1 is the central enzyme of transcription and a major target for the regulation of gene expression. The association of a wide array of accessory proteins with RNAP is critical for the regulation of each phase of the transcription cycle: initiation, elongation, and termination. In addition to accessory factors that interact with RNAP to regulate the transcription process, protein-protein interactions couple RNAP to proteins participating in other cellular processes such as DNA repair (1).
During the development of purification procedures for Escherichia
coli RNAP, we noticed the consistent co-purification of a 110-kDa
polypeptide. The presence of this contaminant through the last step of
varying purification procedures suggested that it was a previously
unidentified factor specifically associated with the RNAP. Here, we
identify the 110-kDa contaminant as the product of the hepA
gene, a putative helicase with extensive sequence similarity to the
SNF2 family of proteins (2-5). Some members of this family, which
contains proteins from viral, prokaryotic, and eukaryotic species, are
DNA-dependent ATPases and participate in cellular processes
such as transcription regulation or DNA repair. We have cloned the
hepA gene and overexpressed and purified the HepA protein.
We show in vitro that RNAP preparations have an ATPase
activity only in the presence of HepA and that HepA binds core RNAP
competitively with the promoter specificity 70 subunit
with a 1:1 stoichiometry and a dissociation constant (Kd) of 75 nM. An E. coli
strain with a disruption in the hepA gene shows sensitivity
to the DNA damaging agent UV light.
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EXPERIMENTAL PROCEDURES |
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Cloning of the hepA Gene-- The hepA gene was PCR amplified from E. coli BL21 (DE3) genomic DNA using the following primers: HEPAleft, 5'-GCCGAACACCCATGGCTTTTACACTTGGTC-3'; HEPAright, 5'-CCATTTTCGGATCCGTTACTGATGCGTTACAACG-3'. NcoI and BamHI sites were engineered into HEPAleft and HEPAright, respectively (underlined), to allow digestion of the amplified products and subsequent ligation into the corresponding sites of the T7-based expression vector pET15b (Novagen) to generate pET15b-HepA. The cloning created a mutation in the second amino acid of the protein (from Pro to Ala) as well as a Gln to Arg mutation of the terminal amino acid.
Purification of Overexpressed HepA--
E. coli BL21
(DE3) cells were transformed with pET15b-HepA, grown to an
A600 of 0.6, and induced by the addition of IPTG
to a final concentration of 1 mM. Induction was allowed to
proceed for 4 h. The cells were then harvested by centrifugation
and stored at 80 °C. The cells were thawed, resuspended in 40 mM Tris-HCl, pH 7.9, 300 mM KCl, 10 mM EDTA, and lysed by sonication. The lysate was spun to
pellet cell debris. The supernatant was collected and
poly(ethyleneimine) (PEI) was slowly added to a final concentration of
0.8% (w/v). The PEI pellet was resuspended in TEGD (10 mM
Tris-HCl, pH 7.9, 0.1 mM EDTA, 5% glycerol, 1 mM DTT) + 1 M NaCl, eluting HepA. The HepA
containing supernatant was then precipitated by slowly adding 35 g
of (NH4)2SO4/100 ml of solution
while stirring, incubating for 15 min, and then centrifuging for 45 min
at 11,000 × g. The pellet was resuspended in 1 ml of TEGD + 0.5 M NaCl, and the sample was loaded onto a Sephacryl S-300
(Amersham Pharmacia Biotech) gel filtration column. The HepA containing
fractions, which were determined by SDS-PAGE and Coomassie staining,
were pooled and precipitated again with
(NH4)2SO4 as described above. The
pellet was resuspended in TEGD and diluted with TEGD until the
conductivity was less than TEGD + 0.2 M NaCl. The sample
was then loaded onto a Poros HQ (PerSeptive Biosystems) anion exchange column equilibrated in TEGD + 0.2 M NaCl. Proteins were
eluted with a linear NaCl gradient from 0.2 to 0.5 M. The
peak of HepA eluted at approximately 0.3 M NaCl. At this
stage, the protein was >95% homogeneous as judged by overloaded
SDS-PAGE and Coomassie staining. Nevertheless, a contaminating DNase
activity was still present, which was effectively removed by gel
filtration chromatography on a Superose 6 HR column (Amersham Pharmacia
Biotech) equilibrated with 40 mM Tris-HCl, pH 7.9, 100 mM KCl, 10 mM MgCl2. Throughout the
purification procedure, protein concentrations were determined using
Bradford reagent (Bio-Rad) and measuring the absorbance at 595 nm.
Native Gel Shift Binding Assays--
50 pmol of HepA protein was
added to 25 pmol of core RNAP in 40 mM Tris-HCl, pH 7.9, 100 mM KCl, 10 mM MgCl2, 5%
glycerol. Samples were left to incubate for 15 min at 37 °C. Xylene
cyanol was added to 0.25% prior to loading on a 5% polyacrylamide
(29.2:0.8 acrylamide:bis) nondenaturing gel (6) in Tris-glycine running buffer, pH 8.3. The gels were electrophoresed at 20-30 mA until the xylene cyanol had migrated about three-fourths the length of the
gel. Protein bands were visualized by Coomassie staining. To
unambiguously identify the protein contents of the visualized bands,
the protein bands of interest were excised from the gel with a scalpel,
crushed, and left to soak for 10 min in SDS loading buffer supplemented
with 5 mM -mercaptoethanol. The samples were then
analyzed by SDS-PAGE and Coomassie staining on a 4-12%
Tris-glycine gradient gel (Novex).
Construction of hepA Disruption Mutants--
Insertion
inactivation mutants of the hepA gene were constructed using
a modified version of a gene replacement method (7) to create a partial
deletion in the hepA locus. The SmaI-digested kanamycin (Km) resistance cassette from pBRkm2 (8) was inserted into
the end-filled PmlI site of pET15b-HepA using standard
molecular biology procedures (9), resulting in a plasmid that carried an interrupted copy of the hepA gene (at codon 304 of 969).
The hepA-Kmr cassette was then excised from
pET15b-HepA with SphI and BspDI and ligated into
the SphI and AccI sites of pMAK705 (7) to construct pSD4. E. coli strains NM522 (Invitrogen) and
JC7623 (recBC sbcBC; Ref. 10) were then transformed with
pSD4 and selected for Km and chloramphenicol (Cm) resistance at
30 °C. Single colonies were transferred to Luria Broth (LB)
containing both Km and Cm and grown at 44 °C until turbid. Dilutions
were spread onto prewarmed plates containing both antibiotics and
incubated at 44 °C to select for cointegrates. The plasmid was then
resolved by growing colonies for one cycle at 30 °C in the presence
of both antibiotics until turbid (overnight) in 100 ml of LB. 100 µl
of turbid culture was then inoculated into 100 ml of LB containing Km
but not Cm, and two cycles of growth were allowed. Single colonies were
then selected on media containing Km and grown at 30 °C.
Cms-Kmr clones were selected by duplicate
plating on media containing both antibiotics. Chromosomal insertion
into hepA was verified by both PCR analysis as well as
Southern analysis.
ATPase Assay--
Highly purified fractions of HepA from the
Superose 6 column were assayed for their ability to hydrolyze the
-phosphate from [
-32P]ATP (11). Reactions (10 µl)
contained 10 µM ATP, 3 µCi of [
-32P]ATP, 40 mM Tris-HCl, pH 7.9, 100 mM KCl, 10 mM MgCl2, and 5 µl of
each protein fraction. The reactions proceeded at 25 °C for 3 h
and were terminated by spotting 1 µl on a PEI-F cellulose plate
(J. T. Baker). The plates were developed in a solution of 0.5 M LiCl, 1 M formic acid and visualized and
quantified using a PhosphorImager. ATPase assays were also performed in
the presence of 0.5 mg/ml tRNA, double-stranded or single-stranded
(boiled) salmon sperm DNA (Life Technologies, Inc.), M13
single-stranded DNA, double-stranded or single-stranded (boiled) pBR322
plasmid DNA, or core RNAP.
UV Sensitivity Assay--
The hepA mutants,
NM522hepA and JC7623
hepA, as well as the
parental strains, NM522 and JC7623, were tested for resistance to UV
exposure as follows. Cells were grown in 10 ml of LB to an optical
density of 0.6-0.7. In a dark room, cells were exposed to a handheld
UV light (254 nM, 4 W, 20-25 cm distance) in 1-min increments up to 6 min. Dilutions were plated, and the percentage of
survival was calculated by comparing colony forming units against control samples not exposed to UV.
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RESULTS |
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A 110-kDa Protein Co-purifies with E. coli RNAP--
The initial
observation that formed the impetus for this study is illustrated in
Fig. 1. In attempting to purify RNAP by a standard procedure (12-14) from E. coli RL324 (harboring a
C-terminal His6 tag in the chromosomal copy of
rpoC, which codes for the RNAP ' subunit, obtained from
R. L. Landick), we noticed nearly stoichiometric amounts of a
contaminating protein with a mobility by SDS-PAGE corresponding to
about 110 kDa. The contaminating protein was present through the final
step of purification in some core RNAP and holoenzyme fractions off an
anion exchange column (Fig. 1, fractions 11, 12,
16, and 17). The contaminant was also present
through the last step of a different purification procedure utilizing
Ni2+ affinity chromatography. The presence of nearly
stoichiometric amounts of the 110-kDa contaminant through the final
steps of two different purification procedures suggested that this
protein interacts with the RNAP itself. The 110-kDa protein was also
present after purification of RNAP from E. coli JC7623 (10),
the parent strain of RL324 containing the wild-type rpoC
gene, eliminating the possibility that the 110-kDa contaminant
adventitiously associated with the RNAP from RL324 through the
His6 tag at the C terminus of the
' subunit.
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The 110-kDa Contaminant Is the Product of the HepA Gene-- The N-terminal sequence of the 110-kDa contaminant was determined to be XPFTLGQRWISDTESELGL. A data base search revealed a single match to the product of an open reading frame denoted hepA (helicase putative; Refs. 15-17). The inferred amino acid sequence of HepA contained amino acid sequence similarity with motifs I, II, and III of the six "DEAD box" helicase motifs (2). Later, a frameshift in the original DNA sequence was postulated that revealed that the downstream sequences also contained DEAD box helicase motifs V and VI (3). Based on extensive sequence similarity, HepA has already been grouped with the SNF2 family of putative helicases (3, 5).
We used PCR methods to clone the hepA gene into a T7-based overexpression vector (18). Because of ambiguity in the HepA sequence between helicase motifs III and V (where the frameshift was proposed to occur but its exact location could not be determined; Ref. 3), we sequenced this region of the gene. The sequencing confirmed the presence of a frameshift in the original sequence. The correct sequence gave rise to a peptide of 969 amino acids and a calculated molecular mass of 109,700 Da, consistent with the SDS-PAGE mobility. Between the time our sequencing was completed and this manuscript was written, an updated sequence of this region of the E. coli genome was submitted to GenBank (accession number AE000116 U00096). This sequence exactly matched our sequence. When induced with IPTG, HepA was expressed to very high levels comprising nearly 50% of total cellular protein. Overexpression of the protein had no obvious toxic effects. Upon cell lysis, the bulk of the overexpressed HepA was found in the soluble fraction. We purified the overexpressed HepA with a procedure similar to that used to purify RNAP (14). In the final step of purification by anion exchange chromatography, the RNAP and HepA bound to the column in buffer containing 0.2 M NaCl. The excess HepA that was not associated with RNAP eluted from the column during a NaCl gradient at about 0.3 M NaCl. The resulting HepA was >95% homogeneous (based on overloaded, Coomassie-stained SDS gels), but in subsequent experiments it became clear that a substantial DNase activity either was associated with HepA or was contaminating it. We therefore performed an additional step of purification, gel filtration over a Superose 6 (Amersham Pharmacia Biotech) column. This effectively removed the DNase activity from the HepA protein.HepA Binds Core RNAP but Not Holoenzyme--
With the highly
purified HepA in hand, we asked whether HepA formed a stable complex
with RNAP in vitro, as suggested above. For this purpose, we
used a native gel shift assay (Fig.
2A). When RNAP holoenzyme was
mixed with a 2-fold molar excess of purified HepA and analyzed by
polyacrylamide gel electrophoresis under nondenaturing conditions,
bands corresponding to free HepA and free RNAP holoenzyme were observed
(Fig. 2A, lane 1). Because mobility on the native
gel is determined by both molecular weight and charge, the band in Fig.
2A corresponding to RNAP holoenzyme could conceivably
contain a holoenzyme-HepA complex with the same mobility as holoenzyme.
Therefore, we confirmed the protein components of the bands labeled in
Fig. 2A by excising them and analyzing their contents by
SDS-PAGE (Fig. 2B). By this method, the band labeled
A in Fig. 2A contains only ',
,
70,
, and
, the components of holoenzyme (Fig.
2B, lane A). Core RNAP alone gave rise to two
bands on the native gel (Fig. 2A, lane 5), likely
because of the presence of core RNAP monomers and dimers in equilibrium
(14). A mixture of HepA and core RNAP (2:1 molar ratio) yielded a band
corresponding to free HepA and a band distinct from the bands observed
for core RNAP alone (Fig. 2A, lane 4, band
C). This distinct band contained
',
,
, and
(the
components of core RNAP) and an apparently stoichiometric amount of
HepA, based on the intensity of the Coomassie stain (Fig.
2B, lane C). Thus, we conclude that HepA forms a
stable complex with core RNAP but not holoenzyme.
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Insertion Inactivation of HepA Results in Sensitivity to DNA Damage-- To assess the role of HepA in cellular processes, we constructed an insertion of a Kmr cassette at codon 304 (between helicase motifs II and III) in the genomic hepA gene of two separate strains of E. coli, NM522, and JC7623 (recBC sbcBC; Ref. 10). Insertion of the Kmr cassette at this position of the hepA gene results in a predicted protein product less than one-third the length of full-length HepA and containing only two of the six helicase motifs (I and II). Thus, the normal function of HepA was undoubtedly disrupted.
The two E. coli strains with the disrupted hepA gene (JC7623
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ATPase Activity Associated with HepA--
Because several members
of the SNF2 family have been shown to be DNA-dependent
ATPases (5), we tested fractions of the highly purified HepA from the
Superose 6 gel filtration column for ATPase activity. Ability to
hydrolyze the -phosphate from [
-32P]ATP was
monitored by thin layer chromatography (11), revealing an ATPase
activity above background (Fig.
4A). The ATPase activity in
each fraction was nearly exactly proportional to the protein concentration in the fraction (Fig. 4B).
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DISCUSSION |
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We have identified HepA, an E. coli protein that shares extensive sequence homology with the SNF2 family of putative helicases (3, 5, 17), as an RNAP-associated factor. We cloned and purified HepA and showed that RNAP preparations have an ATPase activity only in the presence of HepA and that HepA associates with core RNAP in vitro (but not holoenzyme). We disrupted the hepA gene in E. coli, resulting in a phenotype displaying sensitivity to UV exposure
The SNF2 family includes proteins from viral, prokaryotic, and eukaryotic species with roles in cellular processes such as cell cycle control (STH1), transcriptional regulation and chromatin remodeling (ATR-X, BRM, hBRM, MOT1, ISW1, and SNF2), nucleotide excision repair (RAD16 and ERCC6), mitotic recombination (RAD54), and other types of DNA repair (RAD5). The family also includes many proteins with no known function.
HepA is predicted to be an ATPase based on its extensive sequence similarity with other ATPases, and thus the ATPase activity associated with RNAP preparations only in the presence of HepA is likely to belong to HepA itself. The results of our experiments, however, do not rule out the possibility that the ATPase activity is associated with another protein (perhaps the RNAP itself) and that this activity is greatly stimulated by HepA. In contrast to SNF2 family members that have been shown to have ATPase activity (Saccharomyces cerevisiae SNF2 and MOT1, human HIP116A); however, this ATPase activity does not appear to be DNA-dependent.
The close relationship between the SNF2 family of proteins and known helicases (2) led us to test the purified HepA protein for helicase activity on various DNA and RNA substrates. Helicase activity has not been demonstrated for any SNF2 family member, and we were unable to detect any activity for HepA (data not shown).
Because of the observed association between HepA and core RNAP, we also
tested the effect of purified HepA protein on various in
vitro transcription assays. We tested the effect of HepA on abortive initiation (19) by 70 holoenzyme at the T7 A1
promoter. We also formed ternary elongation complexes containing a
20-mer transcript on the T7 A1 tr2 transcription unit (20), added HepA,
and then added nucleotides to initiate transcription elongation. We
then examined the effect of HepA on transcription pausing, on the
overall transcription elongation rate, and on termination at tr2.
Finally, we formed the 20-mer ternary complexes on the T7 A1 tr2
transcription unit and then added ATP and HepA and tested for
displacement of the ternary complexes. In all of these investigations,
we could not observe any effect of the purified HepA protein on
abortive initiation, transcription elongation, or termination or on the
stability of the stalled ternary complexes (data not shown).
We constructed insertion inactivation mutants of hepA to obtain clues to the role HepA plays in cellular processes. The hepA disruption mutants were sensitive to UV exposure, suggesting that they were defective in some DNA repair process. The finding that HepA associates with RNAP links HepA with transcription, whereas the data from the hepA gene disruptions link HepA with DNA repair.
It is interesting to note that the original observation of HepA
co-purification with RNAP came from E. coli RL324 (harboring a C-terminal His6 tag in the chromosomal copy of
rpoC, which codes for the RNAP ' subunit, obtained from
R. L. Landick), and subsequently from E. coli JC7623
(10), the parent strain of RL324 containing the wild-type
rpoC gene, eliminating the possibility that HepA adventitiously associated with the RNAP from RL324 through the His6 tag at the C terminus of the
' subunit. Although we
have found HepA co-purification with RNAP from other E. coli
strains, the amounts of HepA associated with RNAP in these two strains is always substantially greater, suggesting that expression of HepA is
up-regulated in these strains. Both RL324 and its parent JC7623 are
recBC sbcBC mutants. These mutations in the RecBCD enzyme
complex are necessary for the efficient transformation of linear DNA
into E. coli and were thus used for the construction of
RL324. The RecBCD enzyme complex plays important roles in both recombination and DNA repair pathways. Thus, it is interesting to
speculate that the possible disruption of RecBCD function may be
compensated by increased expression of HepA, which we have linked to
DNA repair.
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ACKNOWLEDGEMENT |
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We thank Dr. A. J. Clark for providing the E. coli strain JC7623.
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FOOTNOTES |
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* This work was supported in part by a Burroughs Wellcome Fund Career Award in the Biomedical Sciences (to K. S.) and by grants from the Irma T. Hirschl Trust and a Pew Scholar Award in the Biomedical Sciences (to S. A. D.).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 Rockefeller University Undergraduate Research Fellowships.
¶ To whom correspondence should be addressed: Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-7479; Fax: 212-327-7477; E-mail: darst{at}rockvax.rockefeller.edu.
1 The abbreviations used are: RNAP, RNA polymerase; Cm, chloramphenicol; Km, kanamycin; PEI, poly(ethyleneimine); PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; IPTG, isopropyl-1-thio-b-D-galactopyranoside; DTT, dithiothreitol.
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REFERENCES |
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