Previous Article | Next Article 
Journal of Bacteriology, July 1999, p. 4424-4429, Vol. 181, No. 14
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Methanobacterium thermoautotrophicum RNA
Polymerase and Transcription In Vitro
Trevor J.
Darcy,1
Winfried
Hausner,2
Donald E.
Awery,3
Aled M.
Edwards,3
Michael
Thomm,2 and
John N.
Reeve1,*
Department of Microbiology, The Ohio State
University, Columbus, Ohio 432101;
Institut für Allgemeine Mikrobiologie, Universität
Kiel, 24188 Kiel, Germany2; and Banting
and Best Department of Medical Research, University of Toronto,
Toronto, Ontario M5G 1L6, Canada3
Received 16 February 1999/Accepted 27 April 1999
 |
ABSTRACT |
RNA polymerase (RNAP) purified from Methanobacterium
thermoautotrophicum 
H has been shown to initiate transcription
accurately in vitro from the hmtB archaeal histone promoter
with either native or recombinant forms of the M. thermoautotrophicum TATA-binding protein and transcription factor
TFB. Efforts to obtain transcription initiation from hydrogen-regulated
methane gene promoters were, however, unsuccessful. Two previously
unrecognized archaeal RNAP subunits have been identified, and complex
formation by the M. thermoautotrophicum RNAP and TFB has
been demonstrated.
| |
TEXT |
The biochemistry and molecular
biology of methanogenesis from CO2 and H2 have
been established primarily through studies of Methanobacterium
thermoautotrophicum H and Marburg (26). Several steps in this pathway are catalyzed by isoenzymes or pairs of functionally equivalent enzymes, and the availability of H2
has been shown to determine which of these alternative enzymes are synthesized (19). This regulation occurs at the level of
methane gene transcription; however, the molecular mechanisms by which H2 availability is titrated and communicated
intracellularly into promoter activation or inactivation remain
unknown. As the essential next step in furthering this investigation,
both to understand the regulation of methanogenesis and to determine
how an archaeon senses and signals environmental change and regulates
promoter function, we report here the establishment and
characterization of in vitro transcription systems, using native and
recombinant transcription factors and RNA polymerase (RNAP) from
M. thermoautotrophicum H. Two previously unrecognized
archaeal RNAP subunits have been identified, and complex formation by
M. thermoautotrophicum RNAP and archaeal transcription
factor TFB has been demonstrated.
Purification and identification of subunits of M. thermoautotrophicum H RNAP.
RNAP was purified under
anaerobic conditions from M. thermoautotrophicum cells
resuspended (0.5 g [wet weight]/ml) in TMK buffer (50 mM Tris HCl, 10 mM MgCl2, 50 mM KCl, 20% [vol/vol] glycerol [pH 8]).
The cells were ruptured by passage twice through a French pressure cell
at 20,000 lb/in2, and following centrifugation of the
resulting lysate at 100,000 × g for 1 h at 4°C,
the supernatant obtained was loaded onto a DEAE-cellulose column
(Whatman, Fairfield, N.J.). The column was washed with TMK buffer,
bound proteins were eluted with a linear gradient of 50 to 525 mM KCl
in TMK buffer, and the fractions collected were assayed for nonspecific
transcription activity as previously described (7).
Fractions that contained this activity were combined, diluted with 50 mM Tris HCl (pH 8) to ~75 mM KCl, loaded onto a heparin-Sepharose
column (Pharmacia, Piscataway, N.J.), and washed with TMK buffer, and
the bound proteins were eluted with a 50 mM-to-1 M KCl gradient in TMK
buffer. Fractions with RNAP activity were again combined, diluted with
50 mM Tris HCl to reduce the KCl concentration, loaded onto a Mono-Q
column (Pharmacia), and washed with TMK buffer, and the bound proteins were eluted with a 50 mM-to-1 M KCl gradient in TMK buffer. Fractions that contained RNAP activity were combined and loaded onto a HiLoad 16/60 Superdex 200 gel filtration column (Pharmacia) equilibrated with
TMK buffer containing 300 mM KCl, and the RNAP obtained from this
column was used in promoter-specific in vitro transcription reactions. The polypeptides present in such a M. thermoautotrophicum H RNAP preparation, silver stained
following separation by tricine-sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (tricine-SDS-PAGE), are shown in Fig. 1. Their
identities as specific MTH gene products were determined either
following trypsin digestion by matrix-assisted laser-desorption/ionization-time of flight (MALDI-ToF) mass
spectrometry (3) and/or following transfer to polyvinylidene
difluoride membranes (Bio-Rad Laboratories, Hercules, Calif.) by Edman
degradation and N-terminal amino acid sequencing at the University of
California (Davis, Calif.) protein sequencing facility.
For MALDI-ToF analysis, gel fragments containing the individual
polypeptides were excised and cut into ~1-mm cubes that were washed
with water, dehydrated with acetonitrile, rehydrated in 100 mM ammonium
bicarbonate containing 10 mM dithiothreitol (DTT), and incubated at
50°C for 30 min. The gel fragments were then again dehydrated with
acetonitrile, rehydrated with 100 mM ammonium bicarbonate containing 50 mM iodoacetamide, incubated in the dark for 20 min, washed with
ammonium bicarbonate, dehydrated with acetonitrile, blotted dry, and
rehydrated in 50 mM ammonium bicarbonate containing 5 mM
CaCl2 and 6.25 ng of trypsin/µl (Boehringer Mannheim, Indianapolis, Ind.). Following incubation on ice for 30 min, excess trypsin solution was removed and replaced with 50 mM ammonium bicarbonate containing 5 mM CaCl2, and incubation was
continued at 37°C overnight. Tryptic peptides were eluted from the
gel fragments by two sequential 30-min incubations in 100 mM ammonium
bicarbonate, and the eluates were combined and acetic acid was added to
1%. The peptides were adsorbed onto a C18 reverse-phase
resin, washed with 2% acetonitrile-1% acetic acid, eluted with 65%
acetonitrile-1% acetic acid, and analyzed by MALDI-ToF mass
spectrometry, using 0.5 µl of a matrix solution that contained 20 mg
of 
-cyano-4-hydroxy-trans-cinnamic acid (Sigma, St.
Louis, Mo.) dissolved in 1 ml of 50% acetone-50% isopropanol-1%
acetic acid on a Voyager Elite spectrometer (PerSeptive Biosystems,
Framingham, Mass.) equipped with delayed extraction, a timed ion
selector, and an ion reflector. Data were interpreted with the
assistance of the search engine and nonredundant database maintained by
Zhang and Chait (28).
Transcription in vitro by M. thermoautotrophicum H
RNAP with native and/or recombinant M. thermoautotrophicum
TATA-binding protein (TBP) and TFB.
The template DNA was released
from plasmid pRT74 (25) by DdeI digestion.
Transcription initiated 24 bp downstream from the TATA box element of
the hmtB promoter, resulting in a 193-nucleotide runoff
transcript and extension of a 20-mer primer that hybridized to
hmtB transcripts confirmed that hmtB
transcription initiation occurred at the same site in vivo and in vitro
(Fig. 3A). In vitro transcription reaction mixtures contained (in 100 µl) 20 mM Tris HCl (pH 8), 10 mM MgCl2, 120 mM KCl, 30 µM ATP, 30 µM CTP, 30 µM GTP, 2 µM UTP, 2 mM DTT, 2.2 µCi of
[-32P]UTP (3 kCi/mmol), 1 µg of
DdeI-digested pRT74 DNA (25), 10 µl of RNAP,
and 10 µl of partially purified native TBP (nTBP) and nTFB or 100 ng
of purified recombinant TBP (rTBP) and 600 ng of rTFB. Following
incubation for 30 min at 58°C, the proteins present were removed by
phenol-chloroform extraction, and the RNA products were characterized
by electrophoresis and autoradiography, as previously described
(7, 9).
To obtain nTBP, fractions from the DEAE-cellulose column used to purify
M. thermoautotrophicum RNAP were assayed for activity
in an
in vitro transcription system derived from
Methanococcus thermolithotrophicus that lacked TBP (
7). Active
fractions
were pooled, diluted with 50 mM Tris HCl (pH 8) to ~115 mM
KCl,
and loaded onto a Q-Sepharose ion-exchange column. Unbound
proteins
were eluted with TMK buffer, and bound proteins were then
eluted
with TMK buffer containing a 50 mM-to-1 M gradient of KCl. The
fractions containing TBP activity were pooled and loaded onto
a HiLoad
16/60 Superdex 200 gel filtration column, and the partially
purified
nTBP that eluted from this column in TMK buffer containing
300 mM KCl
was used in this
study.
To obtain nTFB,
M. thermoautotrophicum cells (0.5 g [wet
weight]/ml) were suspended in TK buffer (50 mM Tris HCl [pH 8], 50
mM KCl, 20% [vol/vol] glycerol) and ruptured by passage twice
through a French pressure cell at 20,000 lb/in
2. The
supernatant obtained from this lysate by centrifugation
at
100,000 ×
g for 1 h at 4°C was loaded onto a
phosphocellulose
column (Whatman), unbound proteins were washed from
the column
with TK buffer, and the bound proteins were eluted with a 50 mM-to-1
M gradient of KCl in TK buffer. The fractions that contained
the
partially purified nTFB used in this study were identified by
their
activation of specific transcription when added to reaction
mixtures
that contained
M. thermoautotrophicum RNAP and partially
purified
nTBP.
rTBP and rTFB preparations were generated and purified to establish a
defined in vitro transcription system and to confirm
that the partially
purified nTBP and nTFB preparations contained
these activities. MTH1627
was amplified by PCR from
M. thermoautotrophicum H
genomic DNA by using primers with the sequences
5'-CATTGTCAAGATCGAAAACTGCAGGTT
and
5'-GGGAGGTCTCGAGTTGACAG. The 604-bp product was digested
with
XhoI and
PstI and ligated with
XhoI-plus-
PstI-digested pTrcHisA,
resulting in
plasmid pTD105, which was transformed into
Escherichia coli
Top 10 (Invitrogen, San Diego, Calif.).
Isopropyl-
-
D-thiogalactoside
(IPTG; final concentration,
1 mM) was added to exponentially growing
cultures of
E. coli
Top 10 (pTD105), and after 5 h of incubation
at 37°C, the
E. coli cells were concentrated by centrifugation,
resuspended in a solution containing 50 mM sodium phosphate and
300 mM
NaCl (pH 8), and lysed by passage at 20,000 lb/in
2 through
a French pressure cell. His tag-labeled rTBP was purified
from this
lysate by Ni-nitrilotriacetic acid (NTA) Superflow Ni
2+
affinity chromatography (Qiagen, Chatsworth, Calif.) by following
the
manufacturer's protocol. The same procedure was used to obtain
His-tagged rTFB, except that MTH0885 was PCR amplified from genomic
DNA
using primers with the sequences 5'-GTTCTCTAACCTGCAGAAATTA
and 5'-TGTGGATCCATGGGGGCGAAG, and the resulting 998-bp
product
was digested with
PstI and
BamHI and
cloned into
PstI-plus-
BamHI-digested
pTrcHisA,
resulting in plasmid pTD103. Samples of the purified
recombinant
transcription factors were supplied to ICN Biochemicals
(Cleveland,
Ohio) to obtain rabbit anti-TBP and anti-TFB
antibodies.
Sucrose gradient cosedimentation of M. thermoautotrophicum H RNAP and TFB and immunoprecipitation of
RNAP by anti-TFB antibodies.
Under anaerobic conditions, M. thermoautotrophicum cells resuspended (1 g [wet weight]/ml) in
50 mM Tris HCl (pH 8)-10 mM MgCl2-130 mM KCl were lysed
by passage at 10,000 lb/in2 through a French pressure cell,
and the resulting lysate was centrifuged at 14,000 rpm for 4 min in an
Eppendorf microcentrifuge. An aliquot (500 µl) of the cleared
supernatant was loaded on top of an anoxic 10-ml sucrose gradient (15 to 40% sucrose dissolved in 50 mM Tris HCl [pH 8]-10 mM
MgCl2-130 mM KCl) and centrifuged at 25,000 rpm for
16.5 h at 4°C in a Beckman SW41 rotor. Fractions (500 µl) were
collected and assayed for RNAP activity by measuring poly(dA-dT)
directed [32P]UTP incorporation into trichloroacetic acid
(TCA)-precipitable material and for the presence of TFB and TBP by
Western blotting by using antisera raised against purified rTFB and rTBP.
Immunoprecipitation experiments.
Protein A-Sepharose
preparations lacking antibodies or coupled to anti-TBP or anti-TFB
immunoglobulin G antibodies were mixed and incubated for 3 h at
4°C with aliquots of cleared lysates of M. thermoautotrophicum cells. The matrices were collected by centrifugation, washed four times with TMK buffer containing 0.1% Tween 20 and 2 mM DTT, and then washed with TMK buffer containing 1 M
KCl to disrupt protein-protein complexes. RNAP activity in the 1 M KCl
eluates was measured by assaying poly(dA-dT)-directed incorporation of
[32P]UTP into TCA-precipitable material.
Results. By using assays of poly(dA-dT) transcription
resulting in [
14C]ATP or [
32P]UTP
incorporation into TCA-precipitable material, RNAP preparations
were
isolated from several strains of
M. thermoautotrophicum
during
the 1980s (
1,
13,
24,
27). Consistent with the RNAPs
isolated in parallel from other archaea (
8,
27), these
M. thermoautotrophicum enzymes were reported to contain 8 to
10 subunits
and to more closely resemble eucaryal than bacterial RNAPs,
but
they could not be shown to initiate transcription accurately in
vitro from methanogen promoters. The basis for this deficiency
was
apparently revealed when subsequent studies with other archaeal
in
vitro transcription systems demonstrated that archaeal homologs
of both
the eucaryal TBP and general transcription factor IIB,
designated TFB
in
Archaea, were needed in addition to archaeal
RNAP for
accurate promoter-dependent transcription initiation
(
7,
9,
14,
18). In the
M. thermoautotrophicum H genome
report
(
23), MTH1627 and MTH0885 were annotated as encoding
TBP and
TFB, respectively, and MTH genes encoding RNAP subunits
A', A", B', B",
D, E', E", H, K, L, and N were so designated based
on sequence
similarities to RNAP subunits characterized previously
from other
archaea, primarily from
S. acidocaldarius (
14,
15).
None of the MTH genes, however, encoded amino acid sequences
related
to those reported for
S. acidocaldarius RNAP
subunits F and G
(
15). As illustrated in Fig.
1, RNAP preparations purified from
M. thermoautotrophicum H contained seven polypeptides
that were
readily resolved by tricine-SDS-PAGE and four smaller
polypeptides
that migrated with similar electrophoretic mobilities.
Each of
these polypeptides was identified as the product of a specific
MTH gene by MALDI-ToF analysis of tryptic peptides (
3)
and/or
by Edman degradation and N-terminal amino acid sequencing. The
six largest were confirmed as RNAP subunits A', B', B", A", D,
and E',
encoded as predicted by MTH1051, MTH1050, MTH1049, MTH1052,
MTH0037,
and MTH0264, respectively (
23), but the seventh largest
was
encoded by MTH1324, a gene not previously recognized as encoding
an
RNAP subunit. Similarly, three of the smaller polypeptides
were
confirmed as RNAP subunits H, L, and K, encoded as predicted
by
MTH1048, MTH1317 and MTH0042, respectively, but the fourth
was encoded
by a previously unannotated open reading frame located
between MTH0680
and MTH0681, here designated MTH0680.5 (Fig.
2).
Based on their consistent presence in
near-stoichiometric amounts
and their limited but detectable sequence
similarities to eucaryal
RNAP subunits Rpb4 and Rpb12 (
16),
the MTH1324 and MTH0680.5
gene products have been designated as RNAP
polymerase subunits
F and P, respectively (Fig.
1). Additional
circumstantial support
for this functional designation is provided by
the genomic locations
of MTH1324 and MTH0680.5 (both are directly
downstream and potentially
cotranscribed with ribosomal
protein-encoding genes) and by the
conservation of MTH1324 and
MTH0680.5 homologs in all completed
archaeal genome sequences (Fig.
2)
(
2,
6,
11,
12). The
genomic organization of MTH1324 and
MTH0680.5 homologs, directly
downstream of rpL21- and rpL37a-encoding
genes, respectively,
is also conserved in
Methanococcus
jannaschii,
Archaeoglobus fulgidus,
and
Pyrococcus horikoshii (
2,
11,
12). The MTH1324
homolog
and rpL21-encoding gene are also adjacent in the
Pyrobaculum aerophilum genome but are transcribed
divergently, and although an MTH0680.5
homolog and an rpL37a-encoding
gene are present, they are not
adjacent in this crenarchaeal genome
(Fig.
2) (
6).
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 1.
Identities of M. thermoautotrophicum H
RNAP subunits silver stained after separation by tricine-SDS-PAGE.
Stetter et al. (24) identified eight subunits (designated by
the letters O and A through G) in RNAP preparations from M. thermoautotrophicum Winter. Based on estimated sizes and very
similar patterns of SDS-PAGE resolution, O and A through F appear to
have been homologs of the M. thermoautotrophicum H
subunits here designated A', B', B", A", D, E', and F, and subunit G
was a mixture that contained the four polypeptides designated subunits
H, L, K, and P. With the exception of subunits F and P, the M. thermoautotrophicum H subunits are homologs of subunits
identified previously for Sulfolobus acidocaldarius RNAP
(14, 15). M. thermoautotrophicum H RNAP
preparations did not contain a homolog of the S. acidocaldarius subunit G, and the MTH0265 and MTH0040 gene
products, predicted to be RNAP subunits E" (6.7 kDa) and N (6.4 kDa)
(23), were not detected, although their presence may have
been masked within the cluster of similarly sized, small subunits.
Eucaryal and bacterial homologs of the M. thermoautotrophicum H subunits are listed based on motif
conservation and sequence alignments (5, 16, 21, 22).
Members of the MTH1324 family (subunit F) have limited similarity to
eucaryal Rpb4, a nonessential subunit of RNAPII that in yeast
participates in transcription under stress conditions and at
temperature extremes (4, 20). All members of the MTH0680.5
family (subunit P) contain four cysteinyl residues arranged in a manner
consistent with a C-4 zinc finger motif and homology to Rpb12 (see Fig.
2C). .
|
|
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 2.
Organization and conservation of the rpoF (A)
and rpoP (B) regions in the genomes of M. thermoautotrophicum (MT), A. fulgidus (AF), M. jannaschii (MJ), P. horikoshii (PH), and P. aerophilum (PA) (2, 6, 11, 12, 23). Arrows indicate
directions of transcription, shading patterns show homology between
genes, and broken lines indicate nonadjacent locations. MTH1323 and
MTH0681 and their homologs are predicted to encode rpL21 and rpL37a,
respectively. MTH1325 and MTH0680 gene products and their homologs are
unknown. Amino acid sequences (C) of MTH0680.5 (MT) and MJ0593.5 (MJ)
aligned with their archaeal homologs and the sequences of Rpb12 from
Saccharomyces cerevisiae (SC), Schizosaccharomyces
pombe (SP), and Homo sapiens (HS) (21, 22).
Identical and similar (indicated by asterisks) amino acid residues are
identified in the conserved sequence. Four cysteinyl residues predicted
to form a C-4 type of zinc finger are boxed. The numbers of amino acid
residues present, but not shown, at the N termini of the eucaryal
proteins are indicated by the numbers in parentheses. The PH homolog
may have longer N-terminal sequences initiated 28 codons upstream at an
in-frame ATG (11).
|
|
M. thermoautotrophicum RNAP initiated transcription
accurately in vitro, using templates that carried the promoter for the
M. thermoautotrophicum archaeal histone-encoding gene
hmtB (
25)
when supplied with either partially
purified preparations of nTBP
and nTFB and/or recombinant His-tagged
versions of these archaeal
transcription factors purified from
E. coli (Fig.
3). Primer extension
experiments confirmed that
hmtB transcription initiated at
the
same site, 24 nucleotides downstream from the TATA box element
of
the
hmtB promoter, both in vivo and in vitro, and changing
the TATA box sequence from 5'-TTTATATA to 5'-TTTGGATA
eliminated
transcription initiation in vitro. Accurate
transcription initiation
also occurred in vitro in reaction mixtures
supplied with the
heterologous templates that carried the
tRNA
Val and archaeal histone
hmfB promoters from
Methanococcus vannielii and
Methanothermus
fervidus, respectively, used previously in
archaeal in vitro
transcription systems (
7,
9). Transcription
initiation was
not, however, detected in reaction mixtures supplied
with templates
that carried the
M. thermoautotrophicum
H
2-regulated
mcr,
mrt, and
ftr methane gene promoters (
19) or with templates
that contained the upstream intergenic and coding regions of the
M. thermoautotrophicum TBP and TFB genes (results not
shown).
Based on these observations, it seemed likely that one or more
additional factors were required to activate transcription from
these
promoters, but assays of many fractions obtained from
M. thermoautotrophicum cell lysates by several different
chromatographic
procedures failed to detect such an activating factor.
MTH1314
is predicted to encode a polypeptide related to eucaryal RNAPII
subunit Rpb9 (also designated transcription elongation factor
TFIIS
[
16,
23]), and this polypeptide was not present in the
M. thermoautotrophicum RNAP preparations. MTH1314 was
therefore
PCR amplified, cloned, and expressed in
E. coli;
however, addition
of the recombinant MTH1314 gene product, purified by
His tag affinity
chromatography from
E. coli, also did not
activate transcription
from the H
2-regulated
mcr
methane gene promoter in vitro.
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 3.
(A) Template DNA, obtained by DdeI digestion
of plasmid pRT74 (25), and origin of the 193-nt runoff
transcript. (B) Autoradiogram of in vitro-synthesized transcripts. A
plus sign indicates that a reaction mixture contained the protein
listed. Lane S, size standards; nt, nucleotides.
|
|
Because very large, multicomponent RNAPII holoenzyme complexes are
required for eucaryal transcription activation in vitro
(
10,
16,
17), it seemed possible that transcription initiation
from the
methane gene promoters might be detected if less-purified
RNAP
preparations, in which the RNAP activity remained part of
a larger
complex, were used. Such complexes exhibiting RNAP activity
were
therefore isolated by phosphocellulose chromatography, sucrose
gradient
sedimentation, and immunoprecipitation, but they did
not exhibit
detectable transcription initiation in vitro from
the methane gene
promoters. Considerable effort was made to minimize
the exposure of
cell extracts, complexes, and fractions to air,
but the addition of 1 mM DTT was still needed to obtain transcription
in vitro in reaction
mixtures provided with the
hmtB promoter-containing
templates. In this regard, it is noteworthy that the C-terminal
region
of the subunit D (MTH0037) of
M. thermoautotrophicum RNAP
contains eight cysteinyl residues, consistent with the presence
of a
ferredoxin-like [4Fe-4S] center, and it remains possible
that
transient exposure to oxidizing conditions irreversibly inactivated
the
factor(s) needed for transcription in vitro from other
M. thermoautotrophicum promoters. The ferredoxin-like motif is also
present in subunit D of
A. fulgidus and
S. acidocaldarius RNAPs
(
19a).
Further characterization of the large RNAP-containing complexes led to
the discovery that
M. thermoautotrophicum TFB and RNAP
associate independently of TBP. They cosedimented through sucrose
gradients, apparently within a complex that does not contain TBP
(Fig.
4), and RNAP activity was
immunoprecipitated from
M. thermoautotrophicum cell lysates
by protein A-Sepharose carrying anti-TFB antibodies,
whereas very
little RNAP activity bound to protein A-Sepharose
carrying anti-TBP
antibodies (Fig.
4). RNAP activity was also
removed from
M. thermoautotrophicum cell lysates by affinity to
His-tagged TFB
immobilized on Ni-NTA Superflow, whereas only background
levels of RNAP
activity bound to His-tagged TBP immobilized on
Ni-NTA Superflow
(results not shown).
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 4.
Sucrose gradient cosedimentation of M. thermoautotrophicum H RNAP and TFB and immunoprecipitation of
RNAP by anti-TFB antibodies. Fractions containing TFB and TBP were
identified by Western blotting. The inset shows the results of
immunoprecipitation experiments with protein A-Sepharose preparations
lacking antibodies ( ) or coupled to anti-TBP (-TBP) or anti-TFB
(-TFB). The average values for four separate experiments are shown,
with the RNAP activity eluted from each matrix calculated as a
percentage of the RNAP activity eluted from the matrix carrying
anti-TFB antibodies.
|
|
| |
ACKNOWLEDGMENTS |
This research was supported by grants from the U.S. Department of
Energy (DE-FGO2-87ER13731), the Deutsche Forschungsgemeinschaft, the
Fonds der Chemischen Industrie, the Medical Research Council (MRC) of
Canada, and NATO (collaborative research grant 950733). T.J.D. was
supported by a U.S. Air Force Predoctoral Fellowship, and A.M.E. was
supported as an MRC scientist.
We thank J. Kane for providing the tRNA-expression plasmid pRI952 and
S. Fitz-Gibbon and J. H. Miller for providing Pyrobaculum aerophilum genome data prior to publication.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, The Ohio State University, Columbus, OH 43210. Phone:
(614) 292-2301. Fax: (614) 292-8120. E-mail:
reeve.2{at}osu.edu.
| |
REFERENCES |
| 1.
|
Brown, J. W., and J. N. Reeve.
1989.
Transcription initiation and a RNA polymerase binding site upstream of the purE gene of the archaebacterium Methanobacterium thermoautotrophicum strain H.
FEMS Microbiol. Lett.
60:131-136.
|
| 2.
|
Bult, C. J.,
O. White,
G. J. Olsen,
L. Zhou,
R. D. Fleischmann,
G. G. Sutton,
J. A. Blake,
L. M. FitzGerald,
R. A. Clayton,
J. D. Gocayne,
A. R. Kerlavage,
B. A. Dougherty,
J.-F. Tomb,
M. D. Adams,
C. I. Reich,
R. Overbeek,
E. F. Kirkness,
K. G. Weinstock,
J. M. Merrick,
A. Glodek,
J. L. Scott,
N. S. M. Geoghagen,
J. F. Weidman,
J. L. Fuhrmann,
D. Nguyen,
T. R. Utterback,
J. M. Kelley,
J. D. Peterson,
P. W. Sadow,
M. C. Hanna,
M. D. Cotton,
K. M. Roberts,
M. A. Hurst,
B. P. Kaine,
M. Borodovsky,
H.-P. Klenk,
C. M. Fraser,
H. O. Smith,
C. R. Woese, and J. C. Venter.
1996.
Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii.
Science
273:1058-1073[Abstract].
|
| 3.
|
Courchesne, P. L.,
R. Luethy, and S. D. Patterson.
1997.
Comparison of in-gel and on-membrane digestion methods at low to sub-pmol level for subsequent peptide and fragment-ion mass analysis using matrix-assisted laser-desorption/ionization mass spectrometry.
Electrophoresis
18:369-381[Medline].
|
| 4.
|
Edwards, A. M.,
C. M. Kane,
R. A. Young, and R. D. Kornberg.
1991.
Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro.
J. Biol. Chem.
266:71-75[Abstract/Free Full Text].
|
| 5.
|
Eloranta, J. J.,
A. Kato,
M. S. Teng, and R. O. J. Weinzierl.
1998.
In vitro assembly of an archaeal D-L-N RNA polymerase subunit complex reveals a eukaryote-like structural arrangement.
Nucleic Acids Res.
26:5562-5567[Abstract/Free Full Text].
|
| 6.
| Fitz-Gibbon, S., and J. H. Miller. 1999. Personal communication.
|
| 7.
|
Gohl, H. P.,
B. Gröndahl, and M. Thomm.
1995.
Promoter recognition in archaea is mediated by transcription factors: identification of transcription factor aTFB from Methanococcus thermolithotrophicus as archaeal TATA-binding protein.
Nucleic Acids Res.
23:3837-3841[Abstract/Free Full Text].
|
| 8.
|
Gropp, F.,
W. D. Reiter,
A. Sentenac,
W. Zillig,
R. Schnabel,
M. Thomm, and K. O. Stetter.
1986.
Homologies of components of DNA-dependent RNA polymerases of archaebacteria, eukaryotes and eubacteria.
Syst. Appl. Microbiol.
7:95-101.
|
| 9.
|
Hausner, W.,
J. Wettach,
C. Hethke, and M. Thomm.
1996.
Two transcription factors related with the eucaryal transcription factors TATA-binding protein and transcription factor IIB direct promoter recognition by an archaeal RNA polymerase.
J. Biol. Chem.
271:30144-30148[Abstract/Free Full Text].
|
| 10.
|
Holstege, F. C. P., and R. A. Young.
1999.
Transcriptional regulation: contending with complexity.
Proc. Natl. Acad. Sci. USA
96:2-4[Free Full Text].
|
| 11.
|
Kawarabayasi, Y.,
M. Sawada,
H. Horikawa,
Y. Haikawa,
Y. Hino,
S. Yamamoto,
M. Sekine,
S. Baba,
H. Kosugi,
A. Hosoyama,
Y. Nagai,
M. Sakai,
K. Ogura,
R. Otsuka,
H. Nakazawa,
M. Takamiya,
Y. Ohfuku,
T. Funahashi,
T. Tanaka,
Y. Kudoh,
J. Yamazaki,
N. Kushida,
K. Aohi, and H. Kikuchi.
1998.
Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3.
DNA Res.
5:147-155[Medline].
|
| 12.
|
Klenk, H.-P.,
R. A. Clayton,
J. F. Tomb,
O. White,
K. E. Nelson,
K. A. Ketchum,
R. J. Dodson,
M. Gwinn,
E. K. Hickey,
J. D. Peterson,
D. L. Richardson,
A. R. Kerlavage,
D. E. Graham,
N. C. Krypides,
R. D. Fleischmann,
J. Quackenbush,
N. H. Lee,
G. G. Sutton,
S. Gill,
E. F. Kirkness,
B. A. Dougherty,
K. McKenney,
M. D. Adams,
B. Loftus,
S. Peterson,
C. I. Reich,
L. K. McNeil,
J. H. Badger,
A. Glodek,
L. Zhou,
R. Overbeek,
J. D. Gocayne,
J. F. Weidman,
L. McDonald,
T. Utterback,
M. D. Cotton,
T. Spriggs,
P. Artiach,
B. P. Kaine,
S. M. Sykes,
P. W. Sadow,
K. P. D'Andrea,
C. Bowman,
C. Fujii,
S. A. Garland,
T. M. Mason,
G. J. Olsen,
C. M. Fraser,
H. O. Smith,
C. R. Woese, and J. C. Venter.
1998.
The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.
Nature
390:364-370.
|
| 13.
|
Knaub, S., and A. Klein.
1990.
Specific transcription of cloned Methanobacterium thermoautotrophicum transcription units by homologous RNA polymerase in vitro.
Nucleic Acids Res.
18:1441-1446[Abstract/Free Full Text].
|
| 14.
|
Langer, D.,
J. Hain,
P. Thuriaux, and W. Zillig.
1995.
Transcription in archaea: similarity to that in Eukarya.
Proc. Natl. Acad. Sci. USA
92:5768-5772[Abstract/Free Full Text].
|
| 15.
|
Lanzendörfer, M.,
D. Langer,
J. Hain,
H.-P. Klenk,
I. Holz,
I. Arnold-Ammer, and W. Zillig.
1994.
Structure and function of the DNA-dependent RNA polymerase of Sulfolobus.
Syst. Appl. Microbiol.
16:156-164.
|
| 16.
|
Myer, V. E., and R. A. Young.
1998.
RNA polymerase II holoenzymes and subcomplexes.
J. Biol. Chem.
273:27757-27760[Free Full Text].
|
| 17.
|
Nikolov, D. B., and S. K. Burley.
1997.
RNA polymerase II transcription initiation: a structural view.
Proc. Natl. Acad. Sci. USA
94:15-22[Abstract/Free Full Text].
|
| 18.
|
Qureshi, S. A.,
S. D. Bell, and S. P. Jackson.
1997.
Factor requirements for transcription in the archaeon Sulfolobus shibatae.
EMBO J.
16:2927-2936[Medline].
|
| 19.
|
Reeve, J. N.,
J. Nölling,
R. M. Morgan, and D. R. Smith.
1997.
Methanogenesis: genes, genomes, and who's on first?
J. Bacteriol.
179:5975-5986[Free Full Text].
|
| 19a.
|
Rodriguez-Monge, L., and C. A. Ouzounis.
1998.
A ferrodoxin-like domain in RNA polymerase 30/40-kDa subunits.
Trends Biochem. Sci.
23:169-170[Medline].
|
| 20.
|
Rosenheck, S., and M. Choder.
1998.
Rpb4, a subunit of RNA polymerase II, enables the enzyme to transcribe at temperature extremes in vitro.
J. Bacteriol.
180:6187-6192[Abstract/Free Full Text].
|
| 21.
|
Sakurai, H., and A. Ishihama.
1997.
Gene organization and protein sequence of the small subunits of Schizosaccharomyces pombe RNA polymerase II.
Gene
196:165-174[Medline].
|
| 22.
|
Shpakovski, G. V.,
J. Acker,
M. Wintzerith,
J.-F. Lacroix,
P. Thuriaux, and M. Vigneron.
1995.
Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae.
Mol. Cell. Biol.
15:4702-4710[Abstract].
|
| 23.
|
Smith, D. R.,
L. A. Doucette-Stamm,
C. Deloughery,
H. Lee,
J. Dubois,
T. Aldredge,
R. Bashirzadeh,
D. Blakely,
R. Cook,
K. Gilbert,
D. Harrison,
L. Hoang,
P. Keagle,
W. Lumm,
B. Pothier,
D. Qiu,
R. Spadafora,
R. Vicaire,
Y. Wang,
J. Wierzbowski,
R. Gibson,
N. Jiwani,
A. Caruso,
D. Bush,
H. Safer,
D. Patwell,
S. Prabhakar,
S. McDougall,
G. Shimer,
A. Goyal,
S. Pietrokovski,
G. M. Church,
C. J. Daniels,
J.-I. Mao,
P. Rice,
J. Nölling, and J. N. Reeve.
1997.
Complete genome sequence of Methanobacterium thermoautotrophicum strain H: functional analysis and comparative genomics.
J. Bacteriol.
179:7135-7155[Abstract/Free Full Text].
|
| 24.
|
Stetter, K. O.,
J. Winter, and R. Hartlieb.
1980.
DNA-dependent RNA polymerase of the archaebacterium Methanobacterium thermoautotrophicum.
Zentbl. Bakteriol. Hyg. I Abt. Orig. Teil C
1:201-214.
|
| 25.
|
Tabassum, R.,
K. M. Sandman, and J. N. Reeve.
1992.
HMt, a histone-related protein from Methanobacterium thermoautotrophicum H.
J. Bacteriol.
174:7890-7895[Abstract/Free Full Text].
|
| 26.
|
Thauer, R. K.,
R. Hedderich, and R. Fischer.
1993.
Reactions and enzymes involved in methanogenesis from CO2 and H2, p. 209-252.
In
J. M. Ferry (ed.), Methanogenesis, ecology, physiology, biochemistry and genetics. Chapman and Hall, New York, N.Y.
|
| 27.
|
Thomm, M.,
J. Madon, and K. O. Stetter.
1986.
DNA-dependent RNA polymerases of the three orders of methanogens.
Biol. Chem. Hoppe-Seyler
367:473-481[Medline].
|
| 28.
| Zhang, W., and B. T. Chait. Search engine and
database. [Online.] http://prowl.rochefeller.edu. [4 May 1999, last
date accessed.]
|
Journal of Bacteriology, July 1999, p. 4424-4429, Vol. 181, No. 14
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Zhang, J., Li, E., Olsen, G. J.
(2009). Protein-coding gene promoters in Methanocaldococcus (Methanococcus) jannaschii. Nucleic Acids Res
37: 3588-3601
[Abstract]
[Full Text]
-
Shinzato, N., Enoki, M., Sato, H., Nakamura, K., Matsui, T., Kamagata, Y.
(2008). Specific DNA Binding of a Potential Transcriptional Regulator, Inosine 5'-Monophosphate Dehydrogenase-Related Protein VII, to the Promoter Region of a Methyl Coenzyme M Reductase I-Encoding Operon Retrieved from Methanothermobacter thermautotrophicus Strain {Delta}H. Appl. Environ. Microbiol.
74: 6239-6247
[Abstract]
[Full Text]
-
Micorescu, M., Grunberg, S., Franke, A., Cramer, P., Thomm, M., Bartlett, M.
(2008). Archaeal Transcription: Function of an Alternative Transcription Factor B from Pyrococcus furiosus. J. Bacteriol.
190: 157-167
[Abstract]
[Full Text]
-
Naji, S., Grunberg, S., Thomm, M.
(2007). The RPB7 Orthologue E' Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation. J. Biol. Chem.
282: 11047-11057
[Abstract]
[Full Text]
-
Shin, J.-H., Santangelo, T. J., Xie, Y., Reeve, J. N., Kelman, Z.
(2007). Archaeal Minichromosome Maintenance (MCM) Helicase Can Unwind DNA Bound by Archaeal Histones and Transcription Factors. J. Biol. Chem.
282: 4908-4915
[Abstract]
[Full Text]
-
Kessler, A., Sezonov, G., Guijarro, J. I., Desnoues, N., Rose, T., Delepierre, M., Bell, S. D., Prangishvili, D.
(2006). A novel archaeal regulatory protein, Sta1, activates transcription from viral promoters. Nucleic Acids Res
34: 4837-4845
[Abstract]
[Full Text]
-
Xie, Y., Reeve, J. N.
(2005). Regulation of Tryptophan Operon Expression in the Archaeon Methanothermobacter thermautotrophicus. J. Bacteriol.
187: 6419-6429
[Abstract]
[Full Text]
-
Chen, L., Brugger, K., Skovgaard, M., Redder, P., She, Q., Torarinsson, E., Greve, B., Awayez, M., Zibat, A., Klenk, H.-P., Garrett, R. A.
(2005). The Genome of Sulfolobus acidocaldarius, a Model Organism of the Crenarchaeota. J. Bacteriol.
187: 4992-4999
[Abstract]
[Full Text]
-
Xie, Y., Reeve, J. N.
(2004). Transcription by Methanothermobacter thermautotrophicus RNA Polymerase In Vitro Releases Archaeal Transcription Factor B but Not TATA-Box Binding Protein from the Template DNA. J. Bacteriol.
186: 6306-6310
[Abstract]
[Full Text]
-
Best, A. A., Morrison, H. G., McArthur, A. G., Sogin, M. L., Olsen, G. J.
(2004). Evolution of Eukaryotic Transcription: Insights From the Genome of Giardia lamblia. Genome Res
14: 1537-1547
[Abstract]
[Full Text]
-
Xie, Y., Reeve, J. N.
(2004). Transcription by an Archaeal RNA Polymerase Is Slowed but Not Blocked by an Archaeal Nucleosome. J. Bacteriol.
186: 3492-3498
[Abstract]
[Full Text]
-
Siaut, M., Zaros, C., Levivier, E., Ferri, M.-L., Court, M., Werner, M., Callebaut, I., Thuriaux, P., Sentenac, A., Conesa, C.
(2003). An Rpb4/Rpb7-Like Complex in Yeast RNA Polymerase III Contains the Orthologue of Mammalian CGRP-RCP. Mol. Cell. Biol.
23: 195-205
[Abstract]
[Full Text]
-
Ponting, C. P.
(2002). Novel domains and orthologues of eukaryotic transcription elongation factors. Nucleic Acids Res
30: 3643-3652
[Abstract]
[Full Text]
-
Magill, C. P., Jackson, S. P., Bell, S. D.
(2001). Identification of a Conserved Archaeal RNA Polymerase Subunit Contacted by the Basal Transcription Factor TFB. J. Biol. Chem.
276: 46693-46696
[Abstract]
[Full Text]
-
Briand, J.-F., Navarro, F., Rematier, P., Boschiero, C., Labarre, S., Werner, M., Shpakovski, G. V., Thuriaux, P.
(2001). Partners of Rpb8p, a Small Subunit Shared by Yeast RNA Polymerases I, II, and III. Mol. Cell. Biol.
21: 6056-6065
[Abstract]
[Full Text]
-
She, Q., Singh, R. K., Confalonieri, F., Zivanovic, Y., Allard, G., Awayez, M. J., Chan-Weiher, C. C.-Y., Clausen, I. G., Curtis, B. A., De Moors, A., Erauso, G., Fletcher, C., Gordon, P. M. K., Heikamp-de Jong, I., Jeffries, A. C., Kozera, C. J., Medina, N., Peng, X., Thi-Ngoc, H. P., Redder, P., Schenk, M. E., Theriault, C., Tolstrup, N., Charlebois, R. L., Doolittle, W. F., Duguet, M., Gaasterland, T., Garrett, R. A., Ragan, M. A., Sensen, C. W., Van der Oost, J.
(2001). The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc. Natl. Acad. Sci. USA
10.1073/pnas.141222098v1
[Abstract]
[Full Text]
-
Hanzelka, B. L., Darcy, T. J., Reeve, J. N.
(2001). TFE, an Archaeal Transcription Factor in Methanobacterium thermoautotrophicum Related to Eucaryal Transcription Factor TFIIE{alpha}. J. Bacteriol.
183: 1813-1818
[Abstract]
[Full Text]
-
Werner, F., Eloranta, J. J., Weinzierl, R. O. J.
(2000). Archaeal RNA polymerase subunits F and P are bona fide homologs of eukaryotic RPB4 and RPB12. Nucleic Acids Res
28: 4299-4305
[Abstract]
[Full Text]
-
Todone, F., Weinzierl, R. O. J., Brick, P., Onesti, S.
(2000). Crystal structure of RPB5, a universal eukaryotic RNA polymerase subunit and transcription factor interaction target. Proc. Natl. Acad. Sci. USA
97: 6306-6310
[Abstract]
[Full Text]
-
Mackereth, C. D., Arrowsmith, C. H., Edwards, A. M., McIntosh, L. P.
(2000). Zinc-bundle structure of the essential RNA polymerase subunit RPB10 from Methanobacteriumthermoautotrophicum. Proc. Natl. Acad. Sci. USA
97: 6316-6321
[Abstract]
[Full Text]
-
Macario, A. J. L., Lange, M., Ahring, B. K., De Macario, E. C.
(1999). Stress Genes and Proteins in the Archaea. Microbiol. Mol. Biol. Rev.
63: 923-967
[Abstract]
[Full Text]
-
She, Q., Singh, R. K., Confalonieri, F., Zivanovic, Y., Allard, G., Awayez, M. J., Chan-Weiher, C. C.-Y., Clausen, I. G., Curtis, B. A., De Moors, A., Erauso, G., Fletcher, C., Gordon, P. M. K., Heikamp-de Jong, I., Jeffries, A. C., Kozera, C. J., Medina, N., Peng, X., Thi-Ngoc, H. P., Redder, P., Schenk, M. E., Theriault, C., Tolstrup, N., Charlebois, R. L., Doolittle, W. F., Duguet, M., Gaasterland, T., Garrett, R. A., Ragan, M. A., Sensen, C. W., Van der Oost, J.
(2001). The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc. Natl. Acad. Sci. USA
98: 7835-7840
[Abstract]
[Full Text]
Top
Abstract
Text
