Download Cloning and characterization in Escherichia coli of the gene

FEMS Microbiology Letters 172 (1999) 179^186
Cloning and characterization in Escherichia coli of the gene
encoding the principal sigma factor of an extreme thermophile,
Thermus thermophilus
Makoto Nishiyama a; *, Nobuyuki Kobashi a , Kan Tanaka b , Hideo Takahashi b ,
Masaru Tanokura a
b
a
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Institute of Molecular and Cellular Bioscience, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Received 30 December 1998; received in revised form 19 January 1999; accepted 26 January 1999
Abstract
The nucleotide sequence of the upstream region of the aspartate kinase genes of Thermus thermophilus HB27 revealed the
presence of two open reading frames in the orientation opposite to that of the aspartate kinase genes. The upstream open
reading frame termed ORF375 encodes a protein composed of 375 amino acid residues, possessing amino acid sequence motifs
for methylases. Another open reading frame designated as sigA encodes a protein of 423 amino acid residues which shows
significant identity in amino acid sequence to the principal sigma factor, a component of the DNA-dependent RNA
polymerase holoenzyme. The close proximity of the open reading frames suggested that the two genes are transcribed in a
polycistronic manner. By the use of an Escherichia coli expression system, SigA was produced in a soluble form. An in vitro
transcription assay of purified SigA reconstituted with the core RNA polymerase of E. coli showed that Thermus SigA
functioned as a sigma factor to initiate specific transcription. z 1999 Federation of European Microbiological Societies.
Published by Elsevier Science B.V. All rights reserved.
Keywords : RNA polymerase; Sigma factor ; Thermostable protein; Thermus thermophilus
1. Introduction
Transcription is a major event for gene expression.
RNA polymerase plays the primary role in this process. Although core RNA polymerase of bacteria is
potentially able to elongate RNA chain, speci¢c initiation of transcription requires an additional factor,
a sigma factor, which binds to core RNA polymerase
* Corresponding author. Tel.: +81 (3) 3812-2111;
Fax: +81 (3) 5802-3326.
to form the holoenzyme [1]. The principal sigma factor of Escherichia coli potentiates the transcription of
genes controlled by the consensus promoter consisting of two hexanucleotide sequences, TTGACA and
TATAAT [2] during exponential growth. Sigma factors bind the promoter DNA only in the form of a
complex with core RNA polymerase [3]. In addition
to the role of sigma factors in recognition of promoter elements, sigma factors are directly involved in
DNA melting by binding the non-template DNA
strand in the region around the 310 consensus ele-
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 0 3 9 - 7
FEMSLE 8638 3-3-99
180
M. Nishiyama et al. / FEMS Microbiology Letters 172 (1999) 179^186
ment [4]. In spite of the importance of sigma factors
in the control of gene expression in bacteria, the
mechanisms for promoter recognition, promoter
melting, and promoter clearance are not fully elucidated.
Aspartate kinase is the enzyme which catalyzes the
¢rst step of the reactions in the biosynthetic pathway
leading to threonine, methionine, and lysine [5]. Depending on the availability of nutrition in the environment, the enzyme is inhibited by its endproducts
and/or their intermediates in the pathway [5]. In addition to the inhibition of the enzyme activity, the
endproducts also regulate transcription of the aspartate kinase gene [6]. We cloned the genes encoding
aspartate kinase from Thermus thermophilus (unpublished results) and Thermus £avus [7] and characterized their gene products. During the course of our
studies on the transcriptional control of the genes
encoding aspartate kinase of T. thermophilus, we
found an operon encoding a methylase and a principal sigma factor upstream of the genes coding for
aspartate kinase. This paper describes the cloning
and sequence analysis of the operon, along with
the functional expression of the sigma factor.
2. Materials and methods
2.1. Enzymes and chemicals
Restriction endonucleases, T4 DNA ligase, Taq
polymerase, and the Klenow fragment of DNA polymerase I were purchased from Takara Shuzo (Kyoto, Japan). AmpliTaq Gold was from Perkin Elmer
Japan (Urayasu, Japan). A kit for nucleotide sequencing by the dideoxy chain terminating method
was obtained from Amersham Japan (Tokyo, Japan). Oligonucleotides used were obtained from ESPEC Oligoservice (Tsukuba, Japan). Renaissance, a
kit for non-radioisotope DNA labeling was from
Dupon New England Nuclear (Boston, MA).
2.2. Bacterial strains and plasmids
Escherichia coli JM105 (v(lac pro) thi strA endA
sbcB15 hsdR4 FP traD35 proAB LacIq LacZvM15)
was used as a host for both plasmid construction and
the expression of the sigA gene. E. coli JM105 was
cultured at 37³C in 2UYT medium [8]. T. thermophilus HB27 used as the DNA source for this study
was cultured at 70³C in TM medium [9]. Plasmid
pAKT200 which contains a 1.6-kb PstI fragment
carrying the askAB and a part of the gpt genes of
T. thermophilus HB27 (manuscript in preparation)
was used as a DNA source for chromosome walking.
For expression of the sigA gene in E. coli, plasmid
pTSF101 was constructed as follows. PCR was done
using chromosomal DNA of T. thermophilus as a
template and two synthetic oligonucleotides as primers, 5P-CCGAATTCAAGGAGGTGACATATGAAGAAGAGCAAGCGCAAGAAC-3P and 5P-GGGAAGCTTGGGGGGGCGTCTTCGGGG-3P. The
former was designed to contain an EcoRI site just
upstream of the putative ribosome-binding site for
sigA. This primer was also designed to maximize
translational e¤ciency by replacing the initiation codon, TTG, of the sigA gene with ATG. The latter
was designed to introduce a HindIII site just downstream of the stop codon TAA for sigA. The PCR
conditions were 94³C for 1 min, 50³C for 1 min, and
72³C for 2 min, with a total of 25 cycles. The resulting ampli¢ed DNA was digested with EcoRI and
HindIII, ligated with pUC18 digested with EcoRI
and HindIII, and introduced into E. coli JM105.
The resulting plasmid was named pTSF101. The nucleotide sequence of the DNA fragment ampli¢ed by
PCR was directly con¢rmed by nucleotide sequencing.
2.3. Gene manipulation
For cloning the upstream region of the askAB
gene, the 473 bp BamHI^PstI fragment containing
a 5P portion of the aspartate kinase gene was prepared by agarose gel electrophoresis and used as a
probe for Southern hybridization [10] and colony
hybridization [11]. To clone further upstream (downstream of sigA), the 500-bp BamHI^BglII fragment
containing a central portion of the sigA gene was
prepared in a similar way and used as a probe.
The nucleotide sequence was determined by the dideoxy chain-terminating method using pUC18 or
pUC19 [12]. All restriction sites used for cloning
into pUC plasmids upon sequencing were veri¢ed
by determination of a part of an overlapping sequence.
FEMSLE 8638 3-3-99
M. Nishiyama et al. / FEMS Microbiology Letters 172 (1999) 179^186
2.4. Production and puri¢cation of sigma factor of
T. thermophilus
E. coli cells carrying pTSF101 were precultured at
37³C in 2 ml 2UYT medium supplemented with
50 Wg ampicillin per ml. The preculture was transferred to 200 ml of the fresh 2UYT medium containing 1 mM isopropyl-L-D-thiogalactopyranoside
and 50 Wg ampicillin per ml, and cultured for 15 h
at 37³C. The cells were harvested by centrifugation,
suspended in 10 ml potassium phosphate bu¡er (pH
7.0) containing 5% (w/v) glycerol, and disrupted by
sonication. After centrifugation of the sonicate at
40 000Ug for 30 min, polyethyleneimine (0.01%)
was added to the supernatant to precipitate nucleic
acids in the extract. After similar centrifugation, the
supernatant was heated at 70³C for 30 min and centrifuged again at 40 000Ug for 30 min to remove
heat-labile proteins in E. coli. Protein fractions which
precipitated between 35 and 50% (NH4 )2 SO4 were
collected. The samples were dissolved in 1.5 ml
Tris-HCl (pH 7.5) containing 5% glycerol and applied onto a Mono Q 10/10 anion exchanger column
(Amersham-Pharmacia Japan, Tokyo, Japan). By a
linear gradient of NaCl (0^1.0 M), SigA was puri¢ed
to homogeneity on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [13].
2.5. In vitro transcription assay
The puri¢cation of the E. coli RNA polymerase
core enzyme and the c70 subunit was as described
[14]. The core enzyme was mixed with a 5-fold molar
excess of either c70 or the Thermus SigA, and the
mixtures were incubated for 20 min at 37³C to reconstitute RNA polymerase holoenzymes. The transcription reactions were performed as follows : 3 nM
181
DNA template (pFLAG-CTC, (Eastman)) was incubated with 20 nM RNA polymerase holoenzyme in
35 Wl of transcription bu¡er (20 mM HEPES-KOH,
pH 7.9, 3 mM magnesium acetate, 0.1 mM dithiothreitol, 25 Wg ml31 bovine serum albumin, 100 mM
potassium glutamate) for 20 min at 37³C. Thirteen
microliters of pre-warmed substrate mixture containing 0.53 mM each ATP, GTP, CTP, 0.17 mM UTP,
and 2 WCi [K-32 P]UTP (Amersham; PB 10163) in the
transcription bu¡er was added to allow RNA synthesis, and further incubated for 5 min at 37³C. Two
microliters of heparin (5 Wg Wl31 ) was added to prevent re-initiation, and after 5 min of incubation at
37³C, the reaction was terminated by adding 50 Wl
pre-cooled stop solution containing 40 mM EDTA,
300 Wg ml31 E. coli tRNA at 0³C. Transcripts were
precipitated with ethanol, dissolved in the sample
bu¡er (80% (v/v) formamide, 8 mM EDTA, 0.01%
Bromophenol blue) and examined by denaturing
PAGE containing 8 M urea. Gels were analyzed
with a Bioimage analyzer BAS1000 (Fuji, Tokyo).
3. Results
3.1. Two open reading frames in the upstream
of askAB
In bacteria, a gene encoding a transcriptional regulator is often found in the neighborhood of the gene
under control. The region upstream of the askAB in
pAKT200, however, was too short to encode a protein. We therefore conducted to clone DNA fragment containing further upstream region of askAB.
Southern hybridization using the 473 bp BamHI^
PstI (nt 1^473) fragment as the probe gave a positive
signal of 2.3 kb when T. thermophilus chromosomal
Fig. 1. Restriction maps of the cloned DNA fragments. Restriction maps of pAKT200, pTRD-Bam2.3, and pTRD-GK were shown. The
positions for probes 1 and 2 are also indicated. B, BamHI; G, BglII; K, KpnI ; X, XhoI.
FEMSLE 8638 3-3-99
182
M. Nishiyama et al. / FEMS Microbiology Letters 172 (1999) 179^186
DNA was digested with BamHI. The BamHI fragment was puri¢ed by agarose gel electrophoresis and
inserted into pUC18. Plasmids recovered from ¢ve
transformants showing positive hybridization possessed an identical 2.3-kb insert in either of orientations. One of the plasmids was designated pTRDBam2.3 (Fig. 1).
Nucleotide sequencing of the insert of pTRDBam2.3, however, revealed the absence of a gene
encoding a protein homologous to any transcriptional regulator but showed the presence of two open
reading frames (ORFs) in the direction opposite to
that of askAB. One ORF termed ORF375 encodes a
protein composed of 375 amino acid residues (Fig.
1). The amino acid sequence of ORF375 contains
two methylase motifs; one from 237 to 264 with a
motif, [LIVM]-[LIVMFY]-[DE]-x-G-[STAPV]-G-x[GA]-x-[LIVMF]-[ST]-x(2)-[LIVM]-x(6)-[LIVMY]-x[STAGV]-[LIVMFYHC]-E-x-D, for rRNA adenine
dimethylases and the other from 302 to 308 with a
motif, [LIVMAC]-[LIVFYWA]-x-[DN]-P-P-[FYW],
for an N-6 adenine-speci¢c DNA methylase. Another ORF which started from TTG (nt 1493^1495) as
the translational initiation codon was found within
the cloned region, but was obviously incomplete because of the absence of a stop codon. However, the
truncated ORF was found to encode the NH2 -terminal portion of a protein whose amino acid sequence
showed signi¢cant identity to that of the principal
sigma factor, a component of the DNA-dependent
RNA polymerase holoenzyme. We therefore named
the gene sigA, because the protein actually had sigma
Fig. 2. Amino acid sequence alignment of principal sigma factors. Identical amino acid residues are boxed. TthSigA, SigA of T. thermophilus ; BsuSigA, SigA of B. subtilis [20]; EcoRpoD, RpoD of E. coli [16]; ScoHrdB, HrdB of Streptomyces coelicolor A3(2) [21].
FEMSLE 8638 3-3-99
M. Nishiyama et al. / FEMS Microbiology Letters 172 (1999) 179^186
183
Fig. 3. Production and puri¢cation of SigA from recombinant E. coli cells. Lanes 1, 3, and 5, E. coli JM105 harboring pUC18; lanes 2,
4, and 6, E. coli JM105 carrying pTSF101. Lanes 1 and 2, precipitants of E. coli cell extracts ; lanes 3 and 4, supernatants of E. coli cell
extracts; lanes 5 and 6, supernatants after heat treatment at 70³C for 30 min of the samples shown in lanes 3 and 4, respectively; lanes 7
and 8, molecular weight markers : phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), and carbonic anhydrase (30 kDa) ; lane 9; puri¢ed SigA (full length); lane 10, puri¢ed SigA (truncated).
activity to recruit the core RNA polymerase from E.
coli to a promoter and initiate speci¢c transcription,
as will be described below. Since spacing between the
stop codon TGA of ORF375 and the initiation codon for sigA was only 3 bp, these two genes are
probably transcribed in a polycistronic manner.
To clone the DNA fragment covering the whole
sigA coding region, we re-performed chromosome
walk using the 500-bp BamHI^BglII fragment (nt
1712^2217) containing the central portion of the
sigA gene as probe and cloned a KpnI^BglII fragment of approximately 4.6 kb was cloned into
pUC18. One of the plasmids recovered from two
transformants showing positive hybridization signal
was named pTRD-GK (Fig. 1). We determined the
nucleotide sequence of the BamHI^XhoI region of
3156 bp which covers the above two ORFs. The
nucleotide sequence in the paper has been registered
in EMBL, GenBank, and DDBJ data bases under
accession number AB017014. The deduced amino
acid sequence for SigA revealed that the protein is
composed of 423 amino acid residues (Mr , 48 493).
When the amino acid sequence of SigA was aligned
with those of principal sigma factors from other microorganisms, extensive identity in amino acid sequence was found (Fig. 2). This suggested that the
cloned gene encoded the principal sigma factor of T.
thermophilus.
3.2. Production of SigA in E. coli
To express the sigA gene in E. coli, pTSF101 was
constructed. When the E. coli cells carrying pTSF101
were cultured in the presence of IPTG, a heat-stable
protein with apparent molecular mass of 71 kDa was
found on SDS-PAGE (Fig. 3). We puri¢ed the protein of apparent molecular mass of 71 kDa as described in Section 2. During puri¢cation, however,
the amount of the protein of 71 kDa gradually decreased and di¡erent species with apparent molecular
mass of 55 kDa on SDS-PAGE appeared, suggesting
degradation of the protein. When the NH2 -terminal
amino acid sequences of the two proteins separated
by Mono Q column chromatography (Fig. 3) were
determined by automated Edman degradation, the
protein with apparent molecular mass of 71 kDa
possessed the amino acid sequence of Met-Lys-LysSer-Lys-, which was identical to the 1st to 5th amino
acid residues of SigA, while the other species with
apparent molecular mass of 55 kDa possessed the
sequence of Arg-Lys-Asn-Ala-Gln-, which corresponded to the sequence of the 6th to 10th amino
acid residues of SigA. This showed that lysine-speci¢c protease present in E. coli cells cleaved the peptide bond between Lys-5 and Arg-6 of SigA. We at
present do not know the reason for the slow mobility
FEMSLE 8638 3-3-99
184
M. Nishiyama et al. / FEMS Microbiology Letters 172 (1999) 179^186
to recognize the E. coli promoters on a plasmid, an
in vitro transcription assay was carried out with an
E. coli plasmid, pFLAG-CTC. When E. coli c70 was
used as a control, speci¢c transcriptions for tac and
RNA I were observed (Fig. 4, lane 2). When a similar analysis was carried out with Thermus SigA in
place of E. coli c70 , speci¢c transcriptions which corresponded to tac and RNA I were observed (Fig. 4,
lane 3). This results demonstrated that the Thermus
sigma factor also had an activity to associate with
the core enzyme and initiate speci¢c transcription.
4. Discussion
Fig. 4. In vitro transcription experiments with the reconstituted
RNA polymerase holoenzymes. pFLAG-CTC (5336 bp) containing tac and RNA I promoters were tested by the transcription
assay in vitro. The positions of the tac (348 nt) and the RNA I
(108 nt) transcripts were indicated by arrows. Enzymes used
were : lane 1, E. coli core enzyme ; lane 2, E. coli holoenzyme
containing c70 ; and lane 3, holoenzyme reconstituted with E. coli
core enzyme and Thermus SigA.
of SigA, especially the 71-kDa species, on SDSPAGE.
3.3. In vitro transcription activity
The alignment of the amino acid sequence suggested that SigA encoded the principal sigma factor.
To con¢rm that, we examined the ability of SigA (71
kDa) to initiate transcription in combination with E.
coli core RNA polymerase, because the E. coli core
enzyme is known to associate with principal sigma
factors from various sources and initiate speci¢c
transcription. Since both E. coli and Thermus promoters have the same consensus motifs for 335 and
310 elements [15] and SigA was therefore expected
The close location of the ORF375 and sigA genes
suggested that these genes were transcribed as a single mRNA. To our knowledge, this is the ¢rst instance where the gene for the principal sigma factor
forms an operon with a methylase gene. The amino
acid sequence of the putative methylase had motifs
for adenine-speci¢c methylases, suggesting the possibility that the methylase could play a role in the
control of promoter function of a gene(s) or regulation of translation by methylation of rRNA. In various other microorganisms, the gene encoding the
principal sigma factor is often found in an operon
with rpsU and dnaG coding for the 30S ribosomal
protein S21 and DNA primase, respectively [16],
which are involved in two other important cellular
events, translation and replication. It is therefore interesting to identify the substrate of the methylase
and elucidate the regulation of the Thermus
ORF375-sigA operon.
Crystallographic analysis of a fragment of E. coli
c70 , which contains a part of 1.2 and most of all the
region 2, revealed that the conserved regions 1.2, 2.1,
2.2, 2.3, 2.4 are clustered and closely associated in
the tertiary structure [17]. As shown in Fig. 2, amino
acid residues in the conserved regions are conserved
even in the T. thermophilus SigA protein despite the
di¡erence in their thermostabilities. One exception is
the residue at 440. Genetic studies of E. coli c70 have
shown the involvement of Thr-440 of region 2.4 (in
E. coli c70 numbering) in the interaction with the
base at position 312 (T-12ATAAT in E. coli c70
consensus) [18]. In Thermus SigA the position 248
corresponding to 440 of E. coli c70 was occupied
FEMSLE 8638 3-3-99
M. Nishiyama et al. / FEMS Microbiology Letters 172 (1999) 179^186
by Asn. The fact that Thermus sigma factor initiated
speci¢c transcription suggested that e¡ects of the
substitution at position 248 on the recognition of
310 motif was small for Thermus sigma factor.
The core RNA polymerase of E. coli c70 can initiate transcription in a speci¢c manner only after
interaction with a sigma factor which contains amino
acid residues recognizing the 310 and 335 promoter
elements. The sigma factor itself, however, is not
capable of interacting with promoter DNA without
association with the core RNA polymerase [19]. A
crystallographic study of the fragment of E. coli c70
also suggested that a highly acidic stretch of amino
acid residues from 188th to 209th could occupy the
cleft-like structure containing the residues involved
in 310 element recognition, though structures of
most of the region were not determined due to disorder of this region. Malhotra et al. suggested the
possibility that the high acidity of the patch might be
a reason for the failure of the sigma factor itself to
interact with promoter DNA [17]. As was the case
for many other sigma factors, however, an acidic
patch was absent in the corresponding region of
Thermus SigA. On the contrary, the sigma factor
contains many Glu (20) and Asp (9) residues in the
NH2 -terminal region (residues from 1 to 78), giving
calculated pI value of 3.57 of the segment. We may
assume that the NH2 -terminal acidic segment of
Thermus SigA might inhibit DNA interaction of
this sigma factor.
T. thermophilus can grow at high temperature exceeding 80³C. Therefore, it can be a useful source for
heat-stable enzymes and proteins. In the present
study, we have succeeded in the determination of
the primary structure of one of the most heat-stable
sigma factors and its expression in E. coli, along with
the establishment of the easy puri¢cation procedure
for the protein. As was the case for most of other
proteins in T. thermophilus, SigA was highly stable at
high temperature, which enabled us to easily purify
the wild-type SigA because most of the heat-labile
proteins present in E. coli cells could be easily removed by heat-treatment. Until now, despite the biological importance in transcription, no tertiary structure of sigma factor in full length was determined
and only a structure of a fragment of E. coli c70
was determined [17]. The easy puri¢cation process
and high stability of SigA may become some advant-
185
age to further analyses of the sigma factor including
X-ray crystallography.
Acknowledgments
We are grateful to K. Okamoto (Department of
Applied Biological Chemistry, Graduate School of
Agricultural and Life Sciences, The University of
Tokyo) for her assistance in determining the amino
terminal sequences of SigA.
References
[1] Burgess, R.R., Travers, A.A., Dunn, J.J. and Bautz, E.K.
(1969) Factor stimulating transcription by RNA polymerase.
Nature 221, 43^46.
[2] Ishihama, A. (1988) Promoter selectivity of prokaryotic RNA
polymerases. Trends Genet. 4, 282^286.
[3] Dombroski, A.J., Walter, W.A., Record, M.T., Jr., Siegele,
D.A. and Gross, C.A. (1992) Polypeptides containing highly
conserved regions of transcription initiation factor sigma 70
exhibit speci¢city of binding to promoter DNA. Cell 70, 501^
512.
[4] Buckle, M. and Buc, H. (1989) Fine mapping of DNA singlestranded regions using base-speci¢c chemical probes: study of
an open complex formed between RNA polymerase and the
lac UV5 promoter. Biochemistry 28, 4388^4396.
[5] Cohen, G.N. and Saint-Girons, I. (1987) in: Escherichia coli
and Salmonella typhimurium: Cellular and Molecular Biology
(Neidhardt, F.C., Ingraham, J.L., Low, K.B., Magasanik, B.,
Schaechter, M. and Umbarger, H.E., Eds.), Vol. 1, pp. 429^
444. American Society for Microbiology, Washington, DC.
[6] Richaud, F., Phuc, N.H., Cassan, M. and Patte, J.C. (1980)
Regulation of aspartokinase III synthesis in Escherichia coli:
isolation of mutants containing lysC-lac fusions. J. Bacteriol.
143, 513^515.
[7] Nishiyama, M., Kukimoto, M., Beppu, T. and Horinouchi, S.
(1995) An operon encoding aspartokinase and purine phosphoribosyltransferase in Thermus £avus. Microbiology 141,
1211^1219.
[8] Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33,
103^119.
[9] Koyama, Y., Hoshino, T., Tomizuka, N. and Furukawa, K.
(1986) Genetic transformation of the extreme thermophile
Thermus thermophilus and of other Thermus spp. J. Bacteriol.
166, 338^340.
[10] Southern, E.M. (1975) Detection of speci¢c sequences among
DNA fragments separated by agarose gel electrophoresis.
J. Mol. Biol. 98, 513^517.
[11] Grunstein, M. and Hogness, D.S. (1975) Colony hybridiza-
FEMSLE 8638 3-3-99
186
[12]
[13]
[14]
[15]
[16]
M. Nishiyama et al. / FEMS Microbiology Letters 172 (1999) 179^186
tion: a method for the isolation of cloned DNAs that contain
a speci¢c gene. Proc. Natl. Acad. Sci. USA 72, 248^254.
Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad.
Sci. USA 74, 5463^5467.
Laemmli, U.K. (1970) Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature 227,
680^685.
Igarashi, K. and Ishihama, A. (1991) Bipartite functional map
of the E. coli RNA polymerase alpha subunit: involvement of
the C-terminal region in transcription activation by cAMPCRP. Cell 65, 1015^1022.
Maseda, H. and Hoshino, T. (1995) Screening and analysis of
DNA fragments that show promoter activities in Thermus
thermophilus. FEMS Microbiol. Lett. 128, 127^134.
Burton, Z.F., Gross, C.A., Watanabe, K.K. and Burgess,
R.R. (1983) The operon that encodes the sigma subunit of
RNA polymerase also encodes ribosomal protein S21 and
DNA primase in E. coli K12. Cell 32, 335^349.
[17] Malhotra, A., Severinova, E. and Darst, S.A. (1996) Crystal
structure of a sigma 70 subunit fragment from E. coli RNA
polymerase. Cell 87, 127^136.
[18] Waldburger, C., Gardella, T., Wong, R. and Susskind, M.M.
(1990) Changes in conserved region 2 of Escherichia coli sigma
70 a¡ecting promoter recognition. J. Mol. Biol. 215, 267^276.
[19] Dombroski, A.J., Walter, W.A. and Gross, C.A. (1993) Amino-terminal amino acids modulate sigma-factor DNA-binding
activity. Genes Dev. 7, 2446^2455.
[20] Gitt, M.A., Wang, L.F. and Doi, R.H. (1985) A strong sequence homology exists between the major RNA polymerase
sigma factors of Bacillus subtilis and Escherichia coli. J. Biol.
Chem. 260, 7178^7185.
[21] Shiina, T., Tanaka, K. and Takahashi, H. (1991) Sequence of
hrdB, an essential gene encoding sigma-like transcription factor of Streptomyces coelicolor A3(2): homology to principal
sigma factors. Gene 107, 145^148.
FEMSLE 8638 3-3-99