JP2002335968A - Novel transcription factor, its gene and its binding DNA sequence - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To elucidate the mechanism of transcription in prokaryotic cells and eukaryotic cells, a novel bacterial transcription factor, its gene and its binding DN.
Providing an A sequence. Furthermore, using a gene encoding a transcription factor and a binding sequence, an expression vector is constructed,
To provide an efficient method for producing a useful protein by introducing this into a host. SOLUTION: The structure and function of IS1 which is a bacterial insertion factor are elucidated, the structure and function of Pol III promoter involved in transcription of eukaryotic cells are elucidated, and novel bacteria derived from the sequences of these genes are elucidated. Transcription factors, their genes, and transcription factor binding DNA sequences were generated. Incorporating this novel transcription factor gene and transcription factor binding sequence into an expression vector,
It is introduced into a bacterial host to produce a useful protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a bacterial transcription factor and its gene, and a DNA sequence to which the transcription factor binds. Further, the present invention relates to the production of a bacterial expression vector and a bacterial transformant using the transcription factor gene and the transcription factor binding sequence, and a method for producing a protein using the transformant.

[0002]

2. Description of the Related Art As a first step of gene expression, transcription is performed in which the nucleotide sequence of gene DNA is copied as complementary RNA. The transcription reaction involves an RNA polymerase, and in the case of prokaryotes, generally one type of R
In eukaryotic cells, NA polymerase transcribes RNA polymerases I, II, and III. In order for the correct transcription reaction to occur, a transcription factor is required in addition to the polymerase. In the RNA polymerase III (Pol III) system, three factors, TF-IIIA, TF-IIIB, and TF-IIIC, are known.

[0003] On the other hand, transposable (insertion) elements exist on chromosomes and plasmids of prokaryotes. IS1 is known to be an insertion factor (sequence) that transfers its own DNA fragment from one position to the other on a bacterial plasmid or chromosome (Mobile DNA, 109-162, 1989). IS1
Has two adjacent overlapping open reading frames (translated regions) containing left (IRL) and right (IRR) terminal inverted repeats and insA and B'-insB (Proc. Natl. Acad. . Sci. USA,
75, 615-609 (1978), 79,277-281 (1982); J. Mol. Bio
l., 177, 229-245 (1984)). InsA and B'-insB
Translation of the region is performed by a promoter (pins
L) and appears to occur from a single mRNA transcribed from L) (FIG. 1).

[0004] Pol III is used for 5Sr in eukaryotic cells.
Transcribes genes encoding RNA, tRNA and some viral RNAs (Annu. Rev. Biochm. 57, 8
73-914, 1988). The promoter for Pol III is
Located within the gene itself, it is divided into two regions. The promoter regions before and after are A and B, respectively.
They are called blocks and contain highly conserved sequences (Fig. 3)
(Nature, 294, 626-631, 1981). SINE is a major component of interspersed repetitive DNA, most of which have an intragenic Pol III promoter. S
INE is a retroposon that translocates by an RNA-mediated mechanism (Annu. Rev. Biochem. 55, 631-661, 19).
86). They do not have the genes required for self-amplification. Therefore, it is presumed that enzymes such as reverse transcriptase are borrowed from other sources. Al which is SINE
u sequence (Proc. Natl. Acad. Sci. USA, 81, 5291-5295,
1984) is shown schematically in FIG.

As described above, the bacterial insertion factor IS
Although there are many reports of PolIII, which is involved in transcription of eukaryotic cells, and that of IS1, it has not been reported that IS1 contains a PolIII promoter sequence and that the sequence stimulates transcription in bacteria. . Also, there is no report that the Pol III promoter of Alu, a human gene, also stimulates bacterial transcription. In addition, prokaryotic genes encoding factors that interact with the Pol III promoter have been cloned and identified, and bacterial factors with high homology to eukaryotic Pol III promoter binding proteins have been identified. No report has been made.

[0006]

An object of the present invention is to elucidate the mechanism of transcription in prokaryotic and eukaryotic cells and to provide a novel bacterial transcription factor, its gene and its binding DNA sequence. It is still another object of the present invention to provide an efficient method for producing a useful protein by constructing an expression vector using a gene encoding the transcription factor and a binding sequence and introducing the expression vector into a host.

[0007]

Means for Solving the Problems The present inventors elucidated the structure and function of IS1, which is a bacterial insertion factor, and further elucidated the structure and function of Pol III promoter involved in transcription of eukaryotic cells. The present inventors have discovered a novel bacterial transcription factor, its gene, and its binding DNA sequence, and have completed the present invention. Furthermore, the present invention has constructed a novel bacterial expression system using the transcription factor, its gene, and its binding DNA sequence, and has provided a method for producing a useful protein.

That is, the present invention provides a bacterial insertion factor IS1
And a transcription factor-binding sequence that acts as a cis factor to increase bacterial transcription
(Claim 1) or the transcription factor binding sequence comprises the nucleotide sequence shown in the region from base pair position 76 to position 208 of the bacterial insertion factor IS1.
NA (Claim 2) or a transcription factor binding sequence is the sequence of base pairs 97 to 107 of the bacterial insertion factor IS1;
3. The DNA according to claim 1 or 2, wherein the DNA has a sequence from No. 180 to No. 190 (Claim 3), and the transcription factor binding sequence comprises the nucleotide sequence shown in SEQ ID NO: 1. The DN according to any one of claims 1 to 3,
A (Claim 4) or the transcription factor binding sequence is composed of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1, and increases bacterial transcription. DNA (claim 5) according to claim 1, characterized in that it acts as a cis factor for
DNA comprising a transcription factor binding sequence derived from an A polymerase III promoter-like sequence and acting as a cis factor for increasing bacterial transcription (claim 6)
7. The DNA according to claim 6, wherein the RNA polymerase III promoter is an RNA polymerase III promoter of a repetitive sequence Alu of the human genome.
(Claim 7) The DNA according to claim 7, wherein the Alu sequence is a 3′-α1 Alu sequence located on the 3 ′ side of the human α1-globin gene (Claim 8),
The DNA according to claim 7 or claim 8, wherein the Alu sequence is a sequence containing the base sequence shown in the region from base position 1 to base position 88 of the 3'-α1 Alu fragment.

[0009] In the present invention, the DNA according to any one of claims 6 to 9 (claim 10), wherein the transcription factor binding sequence comprises the nucleotide sequence shown in SEQ ID NO: 2.
Alternatively, the transcription factor binding sequence is composed of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 2, and acts as a cis factor that increases bacterial transcription. The DNA according to claim 6 (claim 11), the DNA comprising a sequence complementary to the DNA sequence according to any one of claims 1 to 11 (claim 12), A DNA encoding a transcription factor that enhances bacterial transcriptional activity, derived from a sequence that is derived from Escherichia coli and has identity to the Escherichia coli F sex factor transfer region; The DNA of claim 13, which encodes the factor ArtA protein (claim 14),
The DNA according to claim 13 or 14, which comprises the base sequence shown in SEQ ID NO: 3 (claim 15),
14. The DNA according to claim 13, which comprises a base sequence in which one or several bases have been deleted, substituted or added in the base sequence shown in SEQ ID NO: 3, and acts as a transcription factor. Item 16) and Claim 13
(17) A DNA comprising a sequence complementary to the DNA sequence of any one of (16) to (16).

[0010] The present invention further provides a transcription factor (claim 18) comprising a protein which binds to the DNA according to any one of claims 1 to 11 and has an activity of increasing bacterial transcription. A transcription factor characterized by that the protein consists of the amino acid sequence shown in SEQ ID NO: 4 (claim 19), or one or several amino acids in the amino acid sequence shown in SEQ ID NO: 4 are deleted, substituted or A transcription factor (claim 20) comprising an added amino acid sequence and a protein having an activity of increasing bacterial transcription;
A transcription factor (claim 21), which is encoded by the DNA according to any one of claims 6 and 6 and has a bacterial transcription activity, and the DNA and / or the DNA according to any one of claims 1 to 11 A bacterial expression vector wherein the DNA according to any one of claims 13 to 16 has been introduced into a vector (claim 22), or the vector has a promoter derived from T5 phage. The bacterial expression vector according to claim 22 (claim 23) or the vector has a DNA encoding a predetermined protein.
A bacterial expression vector according to claim 24 (claim 24) or claim 22.
A bacterial transformant obtained by introducing the bacterial expression vector according to any one of claims to 24 into a bacterial host (claim 2).
5) Alternatively, the bacterial host is Escherichia coli, and the bacterial transformant according to claim 25 (claim 26) or the transformant according to claim 25 or 26 is cultured to obtain a protein. The present invention relates to a method for producing a protein, characterized in that it is produced (claim 27).

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The transcription factor binding sequence of the present invention, that is, a DNA comprising a nucleotide sequence to which a transcription factor binds, is derived from a bacterial insertion factor IS1 gene or a polymerase III promoter and is a cis-factor that increases bacterial transcription. The DNA is not particularly limited as long as it acts as a factor. Examples of the DNA comprising the transcription factor binding sequence derived from the bacterial insertion factor IS1 gene include base pair (bp) position 76 of the bacterial insertion factor IS1 gene. Consisting of a sequence containing the nucleotide sequence shown in the region from No. 208 to No. 208
Alternatively, the sequence from bp position 97 to 107 of the bacterial insertion factor IS1 corresponding to the A block of Alu, which is a repeat sequence of the human genome, and the IS1 bp position from 180 to 190 having high homology to the B block of Alu. No. 1, a DNA having an RNA polymerase III promoter-like sequence, a DNA consisting of the base sequence shown in SEQ ID NO: 1, or a base sequence shown in SEQ ID NO: 1
Alternatively, a DNA consisting of a base sequence in which several bases have been deleted, substituted or added, and acting as a cis factor that increases bacterial transcription can be exemplified. Also,
From the above-described polymerase III promoter, preferably a transcription factor binding sequence derived from the RNA polymerase III promoter of the human genome repetitive sequence Alu, more preferably a 3'-α1 Alu RNA polymerase III promoter located 3 'to the human α1-globin gene. The resulting DNA is bp position 1 of the 3'-α1 Alu fragment.
In the DNA consisting of the base sequence including the base sequence shown in the region from No. 88 to the base sequence, the DNA consisting of the base sequence shown in SEQ ID NO: 2 and the base sequence shown in SEQ ID NO: 2,
Alternatively, a DNA consisting of a base sequence in which several bases have been deleted, substituted or added, and acting as a cis factor that increases bacterial transcription can be exemplified. Further, the DNA of the present invention includes a DNA having a sequence complementary to the DNA comprising the transcription factor binding sequence of the present invention. Based on the DNA sequence information and the like, the DNA of the present invention can be prepared by a known method from a gene library of mammals such as humans, bacteria such as Escherichia coli, or the like, or can be chemically synthesized. it can.

[0012] In addition, a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 or 2 or its complementary sequence, or a nucleotide sequence containing a part or all of these sequences is used as a probe to stringently bind various DNA libraries. By carrying out hybridization under mild conditions and isolating DNA hybridizing to the probe, a part of DNA of IS1 derived from Escherichia coli and A
It is also possible to obtain a DNA that binds to a target transcription factor that is as effective as a part of the lu DNA. D thus obtained
NA is also included in the scope of the present invention. Such DNA of the present invention
For example, hybridization conditions for obtaining are as follows: hybridization at 42 ° C., and a buffer containing 1 × SSC and 0.1% SDS.
Washing at 65 ° C., hybridization at 65 ° C., 0.1 × SSC, 0.1% S
Washing at 65 ° C. with a buffer containing DS can be more preferably mentioned. The factors affecting the stringency of hybridization include:
There are various factors other than the above-mentioned temperature conditions, and those skilled in the art can appropriately combine the various components to achieve the same stringency as the above-described hybridization stringency.

The transcription factors used in the present invention include:
Any protein that binds to the DNA comprising the transcription factor binding sequence of the present invention and has bacterial transcription activity, preferably a protein that binds to the B block of the DNA polymerase III promoter, may be used.
Examples of proteins that bind to the B block include Escherichia coli and Saccharomyces cerevisiae (Accession N
o. YSC138TAU), Shizosscharomyces pombe (Accession
No. SPBC336), Drosophila (Accession No. AE00364)
0), Arabidopsis-1 (Accession No. AC022492), Arab
idopsis-2 (Accession No. AC007843), human (Accessi
on No. HSU02619), a protein that binds to a B block derived from a rat (Accession No. A56011), etc., but is not limited thereto. Examples of such a protein include an ArtA protein derived from Escherichia coli consisting of the amino acid sequence shown in SEQ ID NO: 4 and an amino acid sequence shown in SEQ ID NO: 4 in which one or several amino acids have been deleted, substituted or added. And proteins having bacterial transcription activity can be specifically exemplified, wherein the bacterial transcription activity is R
It refers to the activity of promoting transcription by binding to the B block-like sequence of the NA polymerase III promoter. The above Ar
The present inventors have clarified the function of tA for the first time. Further, the above-mentioned protein can be prepared by a known method based on its DNA sequence information and the like, and its origin is not particularly limited.

Examples of the DNA encoding the transcription factor include a protein that binds to the DNA comprising the transcription factor binding sequence of the present invention and has a bacterial transcription activity, preferably a protein that binds to the B block of the RNA polymerase III promoter. Any DNA may be used as long as it encodes, for example, a DNA encoding ArtA derived from Escherichia coli shown in SEQ ID NO: 4,
In the amino acid sequence shown in the above, DN consisting of an amino acid sequence in which one or several amino acids are mutated, that is, one or several amino acids are deleted, substituted or added, and which encodes a protein having transcription activity
A or a DNA consisting of the base sequence shown in SEQ ID NO: 3
And, in the base sequence shown in SEQ ID NO: 3, one or several bases are composed of a base sequence in which deletion, substitution, or addition is performed, and the translation product thereof is derived from DNA or Escherichia coli, which acts as a transcription factor, Specific examples include DNAs encoding transcription factors that increase bacterial transcriptional activity, which are derived from a sequence having the same identity as the E. coli F element transfer region. Further, as the DNA of the present invention,
DNAs comprising a sequence complementary to the DNA encoding the transcription factor of the present invention are also included. The DNA of the present invention described above can be prepared based on the DNA sequence information and the like, for example, E. coli, Saccha
romyces cerevisiae, Shizosscharomyces pombe, Droso
It can be prepared by a known method from a gene library of phila, Arabidopsis, human, rat, or the like, or can be chemically synthesized.

[0015] Further, various DNAs may be used as probes by using a DNA consisting of the base sequence shown in SEQ ID NO: 3 or a sequence complementary thereto or a sequence containing a part or all of these sequences.
Hybridization is performed on the library under stringent conditions, and DNA that hybridizes to the probe is isolated.
It is also possible to obtain a DNA encoding a protein having a desired transcription activity, such as rtA, which is as effective as a transcription factor binding to the B block of the polymerase III promoter.
The DNA thus obtained is also included in the scope of the present invention.

The bacterial expression vector of the present invention is not particularly limited as long as the DNA comprising the transcription factor binding sequence and / or the DNA encoding the transcription factor is a bacterial expression vector introduced into a plasmid vector. However, any of those capable of expressing the target gene or DNA can be used. Examples of the bacterial expression vector include an expression vector having a promoter upstream of the transcription factor binding sequence of the present invention and a target gene or DNA of interest downstream, and the expression vector further encoding a transcription factor. An expression vector containing DNA,
Specific examples include an expression vector containing a DNA encoding a transcription factor, but are not limited thereto. Examples of the promoter include a T5 phage promoter, an RSV promoter, a trp promoter, a lac promoter, a recA promoter, and a λP promoter.
L promoter, lpp promoter, SPO1 promoter, SPO2 promoter, penP promoter, PHO5 promoter, PGK promoter, GA
P promoter, ADH promoter, SRα promoter, SV40 promoter, LTR promoter, C
Specific examples include MV promoter, HSV-TK promoter and the like.

The vector used for the production of the bacterial expression vector of the present invention includes expression vectors derived from plasmids, chromosomes, episomes, liposomes, and viruses.
For example, a vector derived from a bacterial plasmid such as Escherichia coli, a yeast plasmid, a papovavirus such as SV40, a vector derived from a retrovirus, a bacteriophage derived, a transposon derived and a combination thereof,
Examples include those derived from genetic elements of plasmids and bacteriophages, such as cosmids and phagemids.

[0018] The bacterial transformant of the present invention may be any as long as the bacterial expression vector is introduced into a bacterial host.
(BASIC METHODS IN MOLECULAR BIOLOGY, 1986) and
Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUA
L, 2nd Ed., Cold Spring Harbor Laboratory Press, C
old Spring Harbor, NY, 1989) and methods described in many standard laboratory manuals, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection (t
ransvection), microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading (scrape loadi)
ng), ballistic introduction, infection, etc. And, as host cells, Escherichia coli, Streptomyces, Bacillus subtilis, Streptococcus,
Bacterial prokaryotic cells such as Staphylococcus.

Further, by culturing a bacterial transformant into which the bacterial expression vector containing the target gene or DNA has been introduced, the protein or peptide encoded by the target gene or DNA can be strongly expressed. Useful proteins or peptides can be produced in large quantities. To recover and purify such proteins or peptides from cell cultures, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography,
Known methods including hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography, preferably high performance liquid chromatography, are used. In particular, as a column used for affinity chromatography, for example, a column to which an antibody against the protein or peptide is bound, or when a normal peptide tag is added to the protein or peptide, a substance having an affinity for the peptide tag. A protein or peptide can be obtained by using a column to which is coupled. The above protein or peptide purification method can be applied to peptide synthesis.

[0020]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited to these examples. Example 1 (Effects of IS1 internal region on gene expression by IS1-specific and exogenous promoters) Example 1-1 [Results] Generally, it is considered that the strength of gene expression is inversely proportional to the distance between the promoter and the downstream gene. Have been. But,
IS1 using IS1-lacZ fusion construct
Analysis of the control of the gene expression of (SEQ ID NO: 5) showed that the activity of β-galactosidase in pSAM199 in which lacZ is connected in frame with the insA frame at bp position 75 of IS1 was found at bp position 208 of IS1 at insA. Was 50 times lower than the activity of β-galactosidase in pCM101 (J. Mol. Biol. 208, 567-574, 1989) in which lacZ is bound in frame (FIG. 2). Ins at bp position 233 of IS1
The activity of β-galactosidase in pSAM200 in which lacZ is bound in frame to A was also increased 21-fold compared to pSAM199 (FIG. 2). With these results,
The region at bp positions 76-208 of IS1 was suggested to stimulate the gene expression controlled by pinsL. The DNA sequence of that region is shown in SEQ ID NO: 1 in the sequence listing. In the following, search for assumed functions is performed.

IS controlled by an exogenous promoter
Expression of the 1 gene-lacZ fusion gene was also stimulated by the IS1 internal region. This result was obtained as follows. In pSAM255, pSAM254, and pSAM166, the bp positions 53-75, 53-208 of the IS1 fragment were located immediately downstream of the mutated E. coli phage T5 promoter / lac operator region (pT5 ') having a translation initiation signal.
And 53 to 233 are bound in-frame (FIG. 2). The lacZ of these plasmids, respectively,
InsA is bound in-frame. In the absence of IPTG, β-galactosidase activity in pSAM254 and pSAM166 was 137-fold, 91-fold, respectively, compared to pSAM255.
It increased twice (FIG. 2). Also, in the presence of IPTG,
β-galactosidase activity increased (FIG. 2).

(Presence of Pol III promoter-like sequence in IS1 internal region and its role in transcription)
In the process of studying how the internal region of A stimulates gene expression, IS1 bp positions 97-107b
The p site and the 180 to 190 bp site are each Al
u (SEQ ID NO: 6; Proc. Natlacad. Sci. USA 81, 5291-
5295, 1984) and its consensus sequence A block (Nature 29
4, 626-631, 1981), and the B2 sequence (Nuclacids Res. 1)
0, 7461-7475, 1982) with high homology to the B block (FIG. 3). B2 is a rodent (Nuclacids Re
s. 10, 7461-7475, 1982). We examined whether the Pol III promoter-like sequence of IS1 plays a role in stimulating bacterial gene expression. In pSAM267, a fragment containing the IS1 segment (76-208 bp) contains the E. coli phage T5 promoter / two lac operator regions (pT5 ') (Methods Enzymol. 155, 416-433, 1987).
And lacZ with the translation initiation signal (FIG. 2).

PSAM285 and pSAM299 are identical to pSAM267, but p
SAM285 has TGACs at bp positions 97-111 of IS1.
GGGGTGGTGCG has been replaced with GGATCC, pSAM299,
TTCACTTAC is replaced with GC at bp positions 182-190. PSAM285 and pSA in the absence of IPTG
Β-galactosidase activity in M299 is about one-half that in pSAM267 (FIG. 2). Β-galactosidase activity is reduced even in the presence of IPTG (FIG. 2). These results suggest that sequences well conserved in IS1 function to stimulate lacZ expression. Since the IS1 region in pSAM267, pSAM285 and pSAM299 is not translated, lacZ expression appears to be stimulated at the transcriptional stage.

PSAM296 is located at bp positions 182-1 of IS1.
At 90 the TCACTTAC was replaced with GGATCC, otherwise identical to pSAM267 (Figure 2). By this substitution, a GGATCCACCGC sequence is formed with the ACCGC present at bp positions 191 to 195 of IS1. This sequence is more similar to the consensus B block sequence than the wild-type IS1 sequence, with 8/11 bp homology in pSAM296, pSAM2
67 has 7/11 bp homology. The seventh adenine in the B block remains unchanged in the internal promoter of the eukaryotic tRNA gene, and when the adenine is replaced with cytosine (the cases of pSAM296 and pSAM297 also correspond to adenine to cytosine substitutions). (J. Bio. C
hem. 262, 8500-8507, 1987). In the absence and presence of IPTG, the activity of β-galactosidase in pSAM296 was 1.5 times higher than in pSAM267, respectively, as expected.
And 1.4 times higher (FIG. 2).

Furthermore, the bp position 97 of IS1 of pSAM296
Β-galactosidase activity in pSAM295 made by substituting GGATCC from TGACGGGGTGGTGCG at ~ 111 is p
It decreased compared to SAM296 (FIG. 2). Similarly, a similar substitution in pSAM361 reduced β-galactosidase activity (FIG. 2). All these results support the theory that bacterial transcription is stimulated by the Pol III promoter sequence. PSAM251 and pSAM252 in Figure 2
Are bp positions 144 to 208 and 53 to
140 respectively. The activity of β-galactosidase in these plasmids was determined by pCM101 and pSAM254.
PSAM without IS1 region, but lower than in
It does not decrease to levels in cells containing 199 and pSAM255 (FIG. 2). This is probably due to pSAM251 and pSAM2
This seems to be because 52 has a B block and an A block, respectively (FIG. 2).

(Effect of Alu Pol III Promoter Sequence on Transcription Enhancement in Escherichia coli) Alu Sequence (3'-α1 Alu) Located 3 'of Human α1-Globin Gene
Transcripts have bp positions 4-37 and 70-82
Bp positions 4 to 37 contain an A-block-like sequence, and bp positions 70 to 82 overlap with the B-block-like sequence. (FIGS. 1 and 3) (Proc. Natl. Acad. Sc)
i. USA 81, 5291-5295, 1984). P of 3'-α1Alu
It was examined whether the ol III promoter sequence stimulates bacterial transcription. pSAM351 contains Ba containing the IS1 segment.
It is the same as pSAM295 except that the mHI fragment has been replaced by the 3'-α1 Alu fragment at bp positions 1-88. The DNA sequence at bp positions 1 to 88 is shown in SEQ ID NO: 2 in the sequence listing. pSAM354 and pSAM355 are the same as pSAM351, but contain A and B blocks, respectively.
The Alu site at p positions 1-15 and 76-87 has been deleted. Deletion of the A block resulted in decreased β-galactosidase activity, with 1.6 and 3.5 fold decreases in the absence and presence of IPTG, respectively (FIG. 2). Deletion of the B block also reduced β-galactosidase activity (FIG. 2), but to a lesser extent than deletion of the A block. This is because pSAM355 is p
This is probably due to still having the B block-like sequence from SAM295 (see above). The results obtained with the 5 'part of Alu show that Pol III in bacteria
The cis action of the promoter sequence was confirmed.

(The presence of the Pol III promoter sequence is
Influence on RNA production) To confirm that the Pol III promoter sequence stimulates transcription, lac m was used in a pSAM plasmid-bearing strain using a lacZ probe.
RNA was analyzed by Northern blot (FIG. 4a). pSAM
The band pattern of the plasmid-bearing strain was lac mRNA.
Transcripts and decay were similar to patterns from previous Northern blot analysis (J. Bacteriol. 173, 28-36,
1991, J. Mol. Biol. 226, 581-596, 1992). Using mRNA expanded or fragmented in cells or mRNA chains of various sizes fragmented in the process of RNA purification,
Hybridization was performed with the acZ DNA probe. When the hybridized film was scanned and all the hybridization signals were quantified, the presence of lac mRNA was confirmed. Existed l
When ac mRNA is arranged in ascending order, pSAM351, pSAM2
96, pSAM267, pSAM355, pSAM299, pSAM354, and pSAM285. This result is consistent with the β-galactosidase activities of these plasmids arranged in descending order of abundance (FIG. 2), and a decrease in the amount of lac mRNA resulted in a decrease in β-galactosidase activity. Is shown. In addition, the lambda of pSAM351 and pSAM355
c mRNA was examined for attenuation. Transcription initiation was inhibited with rifampicin, and lac mRNA levels over time were analyzed by Northern blot. In the above two strains,
lac mRNA was also reduced (FIG. 4b), indicating that differences in β-galactosidase production
This is not due to differences in stability.

(Cloning of Gene Encoding Transcription Factor)
Transcription of Pol III-dependent tRNA genes includes TFIIIC
(Science 222, 740-748, 1983). TFIIIC is a multi-subunit protein (J. Biochem. 212, 1-11, 1993) and a B block binding protein (J. Bio. Chem. 275, 31480-31487, 200
0, Proc. Natlacad. Sci. USA 89, 10512-10516, 199
2) having. E. coli polymerase does not bind to the cis element site of IS1 (J. Mol. Biol. 177, 247).
-267, 1984), a TFIIIC-like protein appears to be present in bacteria. To investigate this possibility,
The B block of pSAM299 and pSAM296 was present, or the PstI site immediately upstream of the site where
The c-operator sequence was inserted to construct pSAM363 and pSAM371, respectively (FIG. 5). It was conceivable that the binding of the lacI repressor to the operator and the binding of the transcription factor to the cis element near the operator could be antagonized, resulting in suppression of transcription stimulated by the cis element. .

The E. coli JM109 used as the host strain was la
It has a ^{cI q (Gene 33, 103-119,} 1985). PS with the lac operator inserted in the absence of B block
Β-galactosidase activity in AM363 was reduced 2.1-fold compared to pSAM299 (FIG. 5). This is thought to be due to LacI binding to the lac operator and terminating mRNA elongation at the binding site (Proc.
Natl. Acad. Sci. USA 84, 3199-3203, 1987, J. Mol.
Biol. 267, 548-560, 1997). In the case of pSAM371 in which the lac operator was inserted in the presence of the B block, the activity of β-galactosidase was reduced 5.1-fold as compared to pSAM296 (FIG. 5), and the decrease was larger than in the absence of the B block. This indicates that LacI binds to the lac operator to terminate the elongation of mRNA, and at the same time suppresses the binding of transcription factors to cis elements. The gene encoding the transcription factor was cloned as follows.

First, a fragment containing the IS1-lacZ construct regulated by pT5 'of pSAM371 (FIG. 5) was ligated to pACYC184 (J. B.
acteriol. 134, 1141-1156, 1978)
pSAM372 was produced (FIG. 6). Next, the total DNA of E. coli JM109 was partially cut with PstI, and pUC18 cut with PstI.
Was inserted. Escherichia coli JM109 carrying pSAM372 was transformed using this plasmid. 100 μg / m
Many blue colonies appeared on L agar plates containing 1 l ampicillin, 30 μg / ml chloramphenicol, and 40 μg / ml X-gal, some of which were different from other colonies on the same plate. In comparison, it was dark blue. If the PstI fragment obtained by cloning has a gene encoding a substance that acts on the Pol III promoter and stimulates transcription, the transformant having the PstI fragment will have many β-galactosidases. Was expected to be produced. It was also expected that the binding of LacI to the lac operator in the IS1-lacZ construct of pSAM372 would be suppressed by the binding of the factor to the Pol III promoter sequence, resulting in increased β-galactosidase activity. Some dark blue colonies were selected and further analyzed.

For clarity, the results of analysis of the only plasmid found to contain the transcription factor-encoding gene are shown below. The plasmid (designated pSAM380) had a 3.7 kb clone fragment. β-galactosidase activity was 22.7 in cells with pSAM372.
11138 in cells with units pSAM372 and pSAM380
And the presence of pSAM380 was found to increase activity 491-fold (FIG. 6a). Used here,
JM109 with pSAM372, with pSAM372 and pSAM380
JM109 is, had not lost the F 'factor with the lacI ^q. pSAM370 is identical to pSAM372 except that the IS1 segment has been removed (FIG. 6a). p
The presence of SAM380 increased lacZ expression in pSAM370 by 9.9-fold (FIG. 6a).

This increase was due to pSAM380 in pSAM372
Compared to the increased expression of lacZ due to the presence of This indicates that pSAM380 has a gene encoding a transcription factor. pSAM365 is I
PSAM372 except that the S1 segment has been replaced with the corresponding fragment of pSAM363 with the B block removed.
(FIGS. 5 and 6a). The presence of pSAM380 increased lacZ expression in pSAM365 by 187-fold (FIG. 6a). This increase is due to the pSA in pSAM372
Less compared to the increased expression of lacZ due to the presence of M380, indicating that the bacterial factor interacts with the B block sequence. pSA in pSAM365
The expression of lacZ due to the presence of M380 was
It was strong compared to the effect of increasing the expression of lacZ due to the presence of SAM380 (FIG. 6a). This means that the factor is not only a B block but also an I block containing an A block sequence.
This suggests that it is also acting on another site in the S1 internal region.

(Identification of Pol III Promoter Interacting Factor and Its Gene) In order to identify the transcription factor-encoding gene, first, the nucleotide sequence of 600 bp in the 5 'and 3' terminal regions of the cloned fragment of pSAM380 was determined. These sequences are compared with all the nucleotide sequences in GenBank, and the region encoding the transmissibility of E. coli F sex factor (Microbiol. Rev. 58, 162-210, 1994) (GenBank)
nk accession number: U01159). The 5 'and 3' ends of the cloned fragment are the bp of U01159.
They are located at positions 15418 and 19151, respectively.
There are many open reading frames (ORFs) in the 3.7 kb segment (FIG. 6b). Deletion mutant
Made from pSAM380, pSAM381, pSAM382, pSAM383, pSAM
385 and pSAM386. Bp position 154 of U01159
When 18 is bp position 1, the deletion position is b
p position 1845-3558, 1597-2774, 13
50-3558, 969-3503, and 531-37
34. In order to measure the activity of β-galactosidase, JM109 containing pSAM365 was transformed with these mutants.

PSAM365 and pSAM381, pSAM365 and pSAM382, pS
The activity in cells containing AM365 and pSAM383 is similar to pSAM365 and pSAM3
It was almost at the same level of activity in cells containing 80 (FIG. 6b). In contrast, pSAM365 and pSAM385, and pSAM365
The activity in cells containing pSAM386 and pSAM386 was 7.6-fold and 15-fold lower than in cells containing pSAM365 and pSAM380 (FIG. 6b). pSAM387 contains the fragment from bp positions 531 to 1349, which are pSAM365 and pSAM387.
Β-galactosidase activity in cells containing 87 is similar to pSAM36
5 and the same level of activity in cells containing pSAM380 (FIG. 6b). These results show that bp positions 531 to 134
It can be seen that the gene located at No. 9 is responsible for increasing the expression of lacZ. The nucleotide sequence of the F factor segment in pSAM387 is identical to U01159, where
There was an ORF called the tA gene. artA is
bp positions 1087- in the F factor segment of pSAM380
776 (FIG. 6b). GC content of artA is 3
4%, which is an AT-rich gene. The gene sequence of artA is shown in SEQ ID NO: 3 in the sequence listing, and the amino acid sequence of ArtA protein encoded by the gene is shown in SEQ ID NO: 4. ArtA is said to express the ArtA protein, but its function is not known (J. Bacteriol. 171,
213-221, 1989). pSAM384 (ar) with 4 bp (CCGG) introduced at bp position 974 of the F factor segment of pSAM383
(The tA frame is out-of-frame at the insertion point), the activity of β-galactosidase is 16
It decreased twice (FIG. 6). This confirms that artA expresses a transcription factor. Similar results were obtained in experiments using pSAM372 containing both blocks A and B instead of pSAM365.

(Similarity of Pol III promoter binding proteins) PSI-BLAST program (Nuclacids Res. 25,
3389-3402, 1997), ArtA consisting of 104 amino acids did not show homology at the amino acid level to known or putative proteins. From this study, ArtA is B of Pol III promoter.
Since it was thought to act on the block, ArtA and eukaryotic B block-binding subunits were compared by the following method. Human B block binding subunit (Proc.
Natl. Acad. Sci. USA, 91, 1652-1656, 1994) and its homologs found in rats (Mol. Cell Biol. 14, 3053-3064, 1994) with 76% identity and similarity. The homology was high at 84%, while the yeast (Saccharo
myces cerevisiae) (Proc. Natl. Acad. Sci. USA 89,
10512-10516, 1992) and the yeast (Shizosscharomyces pombe) subunit (J. Bi).
o. Chem. 275, 31480-31487, 2000) had low homology with 19% identity and 37% similarity (Fig. 7a).
No homology between vertebrate and yeast subunits has been discovered to date (J. Bio. Chem.
275, 31480-31487, 2000, Proc. Natl. Acad. Sci. US
A, 91, 1652-1652, 1994, Mol. Cell Biol. 14, 3053-3.
064, 1994), but the present inventor found the relevance as follows. Queries rat and S. pombe subunits
-BLAST survey (Nuclacids Res. 25, 3389)
-3402, 1997) and one Drosophila (Drosophil
a) Putative protein (AE003640; alignment score 1)
088, e-value 0) and two Arabidopsis (Arabidopsis)
s) Putative protein (AC022492 and AC007843; alignment score both 43, e-value both 0.02
9) was identified in each iteration.

Thereafter, the BLAST2 sequence program (Nu
cl. Acids Res. 25, 3389-3402, 1997) was used to directly compare the Drosophila putative protein and the Arabidopsis putative protein with all B block binding subunits. FIG. 7a summarizes the results of the homology survey. For example, the two Arabidopsis proteins show 94% identity (94% similarity) over the entire sequence. Arabidopsis protein (AC02
Amino acid positions 9-717 of 2492) show 19% identity (34% similarity) for both amino acid positions 89-808 of the human subunit and rat amino acid positions 889-807 of the rat subunit. Amino acid positions 19-182 of the two Arabidopsis proteins are found in S. p.
It shows 22% identity (39% similarity) for amino acid positions 19-205 of the ombe subunit.
And two Arabidopsis proteins (AC022492 and
AC007843) at each amino acid position 1815-184
9 and 1715-1749 show 44% identity (58% similarity) for amino acid positions 1986-2019 of the human subunit. Drosophila proteins show similarity for almost the entire rat subunit. Amino acid positions 1572-1848 and 1573-1 of Drosophila protein
The C-terminal region of 848 has 19% identity (37% similarity) and 20% with the C-terminal region of amino acid positions 1580-1867 and 1498-1767, respectively, of the Arabidopsis thaliana proteins (AC022492 and AC007843), respectively.
7 (39% similarity) (FIG. 7)
a). These results indicate that vertebrate subunits are related to yeast subunits at the amino acid level,
This shows that the similarity is particularly high between the N-terminal and the C-terminal. Similarly, in the alignment of eukaryotic subunits and their homologs using the CLUSTL W program, the N-terminal and C-terminal short regions showed relatively high homology (Fig. 7b).

Amino acid positions 1-68 and 1037-1110 of the B block binding subunit of S. cerevisiae
Indicates similarity to the HMG box (Proc. Natl. Acad. Sci. USA 89, 10512-10516, 199).
2). The HMG box is a eukaryotic DNA binding motif (70 to 80 amino acids in size) and has been found in various proteins, including transcription factors (FIG. 7b) (Bian
chi, ME in DNA-Protein: Structural Interacti
on 177-200 (ed.Lilley DMJ) (IRL Pressat Oxf
ord University Press, Oxford, UK, 1995)). Interestingly, the HMG box site in S. cerevisiae partially overlaps the conserved site of the B block binding subunit and its homologs. In fact, there was a correspondence between the HMG box alignment and the transcription factor alignment (FIG. 7b). Although the HMG box is diverse, some residues are conserved (Nucl. Acids Re
s. 212493-2501, 1993) (Figure 7b). L of HMG box
The letter-shaped tertiary structure is characterized by conserved, mainly aromatic residues. Aromatic residues are also conserved at the N- and C-termini of the B-block binding subunit and its homologues (FIG. 7b). This strongly suggests that the conserved site for the B block binding subunit and its homologue is the HMG box.

Finally, the conserved sites of the ArtA sequence, the B block binding subunit and its homolog were compared. Ar
The amino acid positions 37-97 and 60-94 of tA can be added to the eukaryotic alignment without altering the alignment (FIG. 7b), and several residues of ArtA are replaced by the same column. In all or most of the eukaryotic proteins (FIG. 7b). Furthermore, W at amino acid positions 62 and 83 of ArtA and I at amino acid position 58 coincide with residues conserved in the HMG box (FIG. 7b). These results indicate that ArtA is associated with eukaryotic B block binding proteins and that ArtA
It turns out to be the first bacterial protein found to contain the MG box.

Example 1-2 [Material and Method Used in Example 1 of the Present Invention] (Construction of plasmid) In order to construct plasmid pSAM199, 5 'of IS1 (GenBank accession number: V00609) was carried out from plasmid pSEK17. The EcoRI-PvuII fragment containing the fragment was digested with EcoRI and SmaI pBluescript
It was cloned in KS + (Stratagene). IS
The 5 'portion of 1 was isolated with an EcoRI-BamHI fragment,
The fragment was replaced with EcoRI-BamHI of pCM101. Construction of the pSAM166 plasmid was performed as follows. The fragment of the lac operator site adjacent to the -35 site in pT5 'was cloned from pQE70 (Qiagen) by PCR.
Amplified by

The following oligonucleotides, BamHI site and p
5'-AATGGAT containing a site corresponding to 47 to 32 bp of QE70
CCATTGTTATCCGCTCAC-3 '(SEQ ID NO: 7: P1) and pQE70
5'-CCACATAGCAGAACTT-3 'corresponding to 82 to 97 bp of
(SEQ ID NO: 8: P2) was used for PCR. In addition, the translation initiation signal and the adjacent fragment of insA were expressed in pSAM168 (J. Mol. Biol. 267, 548-560, 199).
From 7), it was amplified by PCR. BamHI site and 82 of pQE70
5'-TGTGGATCCACAC containing site corresponding to ~ 97 bp site
AGAATTCATTA-3 '(SEQ ID NO: 9: P3) and 5' corresponding to the 200 to 185 bp site of IS1 shown in SEQ ID NO: 5
An oligonucleotide of -GAGAAGCGGTGTAAGT-3 'was used for PCR. PCR products were digested with BamHI and mixed. This mixture was treated with phage T4 ligase and digested with PstI and XmnI. The XmnI-PstI fragment of interest is pSAM185 (J. Mol. Biol. 267, 548-560, 199).
7) was replaced with the XmnI-PstI site.

The plasmid into which the 5 'portion of Alu was introduced was constructed as follows. 5'-CTCACGGATCC-3 'and sequence 5'-
GGATCCATGCC 1 to 87 of the sequence of 3'α1 Alu between 3 '
Two oligonucleotides with each strand at the bp site were applied to Applied Biosystems DNA Synthesizer Model 3
92 and chemically purified on a 15% polyacrylamide / urea gel. These two oligonucleotides were mixed in equal moles (160 pmol) and heated in TE buffer to a total volume of 80 μl at 100 ° C. for 3 minutes. The mixture was gradually cooled at room temperature and left on ice for 1 hour. The annealed DNA sample was digested with BamHI and cloned with BamHI digested pSAM295 to create pSAM351. pSAM363 and pSAM371
Was constructed as follows. 5'-CAATTGTGAGCGGATAACAATTGTGCA-3 'containing lac operator (SEQ ID NO: 10: P4), and further 5'-CAATTGTTATCCGCTCACAATTG
Two oligonucleotides of TGCA-3 '(SEQ ID NO: 11: P5) were chemically synthesized. After these oligonucleotides are mixed and annealed, the DNA sample is
It was cloned with pSAM299 and pSAM296 digested with PstI. The nucleotide sequence of the plasmid constructed above was determined.

The construction of another plasmid was carried out as follows. pCM101 and pSAM199, pSAM200 and pSAM251 are each a 316 bp DNA fragment derived from pJL3, and EcoBR of pBR322.
It has a 25 bp nucleotide from pSEK17 between the I site and the left end of IS1 (Proc. Natl. Acad. Sci. USA
86, 4609-4613, 1989) (Cell, 34, 135-142, 1983).
pSAM199, pSAM255, pCM101, pSAM251, pSAM254, and pS
AM361 is 2 from IS1 fragment and lacZ, respectively.
It contains 5'-GGGGGATC-3 'and 5'-GGGGGATC-3' between 3 bp.
In pSAM200, pSAM252 and pSAM166, lacZ from bp position 23 is directly linked to the IS1 fragment. pCM101, pSAM199, pSAM200 and pSAM251 are derivatives of pBR322. Another pSAM plasmid used in the present invention is constructed using pQE70 as a vector. In the pSAM267 shown in FIG. 2 and the plasmid shown below pSAM267, 5′-CC-3 ′ between the EcoRI site and the 76 bp site of IS1, the 208 bp site of IS1 and another EcoR site.
5'-GGGTACCGAGCTC-3 'exists between the I site.

(Measurement of β-galactosidase activity) pCM1
01 and E. coli JM109 carrying the pSAM plasmid
A medium without PTG (J. Mol. Biol. 240, 52-65, 199)
In 4), the cells were cultured at 37 ° C overnight. The culture medium of JM109 was subcultured to a new medium with and without IPTG, and the OD600 was increased.
Was grown to a value of 0.5, and the β-galactosidase product was published (J. Mol. Biol. 240, 52-65, 199).
It was measured by the method described in 4).

(RNA Analysis) An overnight culture of E. coli JM109 cells having pQE70 and pSAM plasmid
Rich Broth medium (J. Mol. Biol. 196, 445-455,
1987) and cultured at 37 ° C. until the OD600 reached a value of 0.3. IPT so that the final concentration is 2 mM
G was added to the culture and the culture was continued for 2 hours. Total RNA was extracted by the method described in the literature (J. Biol. Chem. 256, 11905-11910, 1981). RNA samples (4 μg) were electrophoresed on a 1.2% agarose gel in the presence of formaldehyde and blotted onto a nylon membrane (Biodyne; PALL). 0.5 kb at the 5 'end of lacZ, labeled using random primer fluorescein labeling kit (NEN) with anti-fluorescein-HRP
The nylon membrane was probed at 60 ° C. with the fragments (81-586 bp site of GenBank ECLACZ).
Probing and washing conditions should be as specified in the kit instructions, and should be enhanced luminol (Nuclei Aci
d Chemiluminescence Reagent, NEN) was used to detect high signals. In order to examine the stability of lac mRNA, rifampicin was added to a culture solution of E. coli JM109 having pSAM351 and pSAM355 cultured for 2 hours in the presence of 2 mM IPTG to a final concentration of 400 μg / ml. At the appropriate time, cells were collected and total RNA was extracted. RNA was transferred to a nylon membrane and hybridized as described above.

Example 2 (Introduction of Transcription Factor Gene and Production of Protein) A gene obtained by ligating a β-galactosidase gene lacZ downstream of a promoter derived from T5 phage was cloned into a plasmid pACYC184. This plasmid was introduced into E. coli K12 JM109. At this time, the β-galactosidase activity was 85 units. On the other hand, the transcription factor gene described in SEQ ID NO: 3 in the sequence listing was cloned together with its unique promoter into plasmid pUC18. This plasmid was further introduced into E. coli into which pACYC184 having lacZ had been introduced. The activity of β-galactosidase of this strain was 850 units. These results indicate that transcription factors increased protein production 10-fold.

Example 3 (Introduction of Transcription Factor Binding Sequence and Production of Protein) A DNA sequence on which a transcription factor acts downstream of a T5 phage-derived promoter (a transcription factor binding sequence having the sequence of SEQ ID NO: 1 in the Sequence Listing) Was ligated, and the lacZ gene was ligated to the end, and cloned into plasmid pACYC184. This plasmid was introduced into E. coli K12 JM109. At this time, the activity of β-galactosidase is
There were 23 units. The plasmid pUC18 in which the gene for the transcription factor was cloned was further introduced into this Escherichia coli (see Example 2). The β-galactosidase activity of this strain was 11138. These results indicate that the presence of the transcription factor and its site of action reduced protein production by 4%.
This indicates that the number has increased 91 times.

[0047]

According to the present invention, a bacterial expression vector into which a DNA encoding the transcription factor of the present invention and / or a DNA comprising the transcription factor binding sequence of the present invention has been introduced can be used as a useful protein by bacteria such as Escherichia coli. When used for the production of, a gene encoding a useful protein can be strongly expressed, and the amount of protein produced can be increased.

[0048]

[Sequence List] SEQUENCE LISTING <110> JAPAN SCIENCE AND TECHNOLOGY CORPORATION National Institute of Health Sciences <120> Novel transcription factors, their genes, and DNA sequences binding to the transcription factors <130> 13-42 <140> <141> < 160> 11 <170> PatentIn Ver. 2.1 <210> 1 <211> 133 <212> DNA <213> Escherichia coli <400> > 2 <211> 88 <212> DNA <213> Homo sapiens <400> 2 ggccgggcgc ggtggctcac acctgtaatt gcagcacttt gccaggctta ggcaggtgga 60 tcacctgaag tcaggggttc gagaccag 88 <210> 3 <211> 501 <212> DNA coli213 <221> CDS <222> (114) .. (425) <400> 3 ccggggtaaa gagaatctgc gtttacttat catgttaaac tccggttatt atttacatga 60 gagtattcat gtctgtaata ttcgcgtttg tgtgatatta atcagaggta caa atg 116 Met 1 gaa ag c aga ga t ag ga t ag ga t ag ga t ag ga t ga t ga t ga t ga t ata ata 164 Glu Lys Arg Ser Phe Lys Glu Lys Leu Glu Ile Ile Arg Asn Ile Ile 5 10 15 aga gag tcg tta ctg gga aat gcg gca att att gcg ctg ata tat gca 212 Arg Glu Ser Leu Leu Gly Asn Ala Ala Ile Ile Ala Leu Ile Tyr Ala 20 25 gca tca cat tca tta ccg gtc aat gct ttc cct gac tat ctt gta ata 260 Ala Ser His Ser Leu Pro Val Asn Ala Phe Pro Asp Tyr Leu Val Ile 35 40 45 agt tta tta agt atc gca gca ggc atc gtt gta tta tgg tta ttt tca 308 Ser Leu Leu Ser Ile Ala Ala Gly Ile Val Val Leu Trp Leu Phe Ser 50 55 60 65 att atc tat att tat ttt tgt gag cta ttc aga agc cac tgg att gcg 356 Ile Ile Tyr Ile Tyr Phe Cys Glu Leu Phe Arg Ser His Trp Ile Ala 70 75 80 gta tgg ttt atc ata tgg tct tca gta atc aac ctc att att ttg tat 404 Val Trp Phe Ile Ile Trp Ser Ser Val Ile Asn Leu Ile Ile Leu Tyr 85 90 95 ggt ttt tat gac cgt ttt ata tgacttagcc tgtcataagc ccccttataa 455 Gly Phe Tyr Asp Arg Phe Ile 100 aaaaccacat cactccggac cataccgatg acaacataat acagat 501 <210> 4 <211> 104 <212> PRT <213> Escherichia coli <400> 4 Met Glu Lys Arg Ser Glu Lys Leu Glu Ile Ile Arg Asn Ile 1 5 10 15 Ile Arg Glu Ser Leu Leu Gly Asn Ala Ala Ile Ile Ala Leu Ile Tyr 20 25 30 Ala Ala Ser His Ser Leu Pro Val Asn Ala Phe Pro Asp Tyr Leu Val 35 40 45 Ile Ser Leu Leu Ser Ile Ala Ala Gly Ile Val Val Leu Trp Leu Phe 50 55 60 Ser Ile Ile Tyr Ile Tyr Phe Cys Glu Leu Phe Arg Ser His Trp Ile 65 70 75 80 Ala Val Trp Phe Ile Ile Trp Ser Ser Val Ile Asn Leu Ile Ile Leu 85 90 95 Tyr Gly Phe Tyr Asp Arg Phe Ile 100 <210> 5 <211> 768 <212> DNA <213> Escherichia coli <400> ggggtggtgc gtaacggcaa 120 aagcaccgcc ggacatcagc gctatctctg ctctcactgc cgtaaaacat ggcaactgca 180 gttcacttac accgcttctc aacccggtac gcaccagaaa atcattgata tggccatgaa 240 tggcgttgga tgccgggcaa ccgcccgcat tatgggcgtt ggcctcaaca cgattttccg 300 ccatttaaaa aactcaggcc gcagtcggta acctcgcgca tacagccggg cagtgacgtc 360 atcgtctgcg cggaaatgga cgaacagtgg ggatacgtcg gggctaaatc gcgccagcgc 420 tggctgtttt acgcgtatga caggctccgg aagacggttg ttgcgcacgt attcggtgaa 480 cgcactatgg cgacgctggg gcgtcttatg agcctgctgt caccctttga cgtggtgata 540 tggatgacgg atggctggcc gctgtatgaa tcccgcctga agggaaagct gcacgtaatc 600 agcaagcgat atacgcagcg aattgagcgg cataacctga atctgaggca gcacctggca 660 cggctgggac ggaagtcgct gtcgttctca aaatcggtgg agctgcatga caaagtcatc 720 gggcattatc tgaacataaa acactatcaa taagttggag tcattacc 768 <210> 6 <211> 94 <212> DNA <213> Homo sapiens <400> 6 aaaaattggc cgggcgcggt ggctcacacc tgtaattgca gcactttgcc aggcttaggc 60 aggtggatca cctgaagtca ggggttcgag acca 94 <210> 7 <211> 25 <212> DNA <213> Artificial Artificial Sequence: P1 <400> 7 aatggatcca ttgttatccg ctcac 25 <210> 8 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P2 <400> 8 ccacatagca gaactt 16 <210 > 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P3 <400> 9 tgtggatcca cac agaattc atta 24 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P4 <400> 10 caattgtgag cggataacaa ttgtgca 27 <210> 11 <211> 27 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P5 <400> 11 caattgttat ccgctcacaa ttgtgca 27

[Brief description of the drawings]

FIG. 1 is a diagram showing the structure of the IS1 gene and Alu gene of the present invention.

FIG. 2. IS1 insertion sequence and Alu in lacZ expression.
FIG. 5 is a diagram showing the effect of the presence of the 5 ′ portion of FIG.

FIG. 3 is a view showing a sequence having homology to a consensus sequence of a Pol III promoter.

FIG. 4. La in strains harboring pQE70 and pSAM plasmids
FIG. 4 shows Northern blot analysis of cmRNA.

FIG. 5: La close to B block in lacZ expression
It is a figure which shows the effect of c operator insertion.

FIG. 6 shows the effect of the presence of the F factor segment on the Pol III promoter that stimulates lacZ expression.

FIG. 7 shows a comparison of the amino acid sequences of B block binding subunits and their homologs.

Continued on the front page F term (reference) 4B024 AA20 BA80 CA04 DA06 EA03 EA04 FA02 GA11 GA19 4B064 AG01 CA02 CA19 CC24 4B065 AA26X AB01 AC14 BA02 CA24 CA31 4H045 AA10 AA20 BA10 CA40 FA74

Claims (27)

[Claims] 1. A DNA comprising a transcription factor binding sequence derived from a bacterial insertion factor IS1 and acting as a cis factor for increasing bacterial transcription. 2. The method according to claim 1, wherein the transcription factor binding sequence is a bacterial insertion factor I.
2. The DN according to claim 1, comprising a base sequence shown in the region from base pair positions 76 to 208 of S1.
A. 3. The method according to claim 1, wherein the transcription factor binding sequence is a bacterial insertion factor I.
A sequence from base pair positions 97 to 107 of S1;
The DNA according to claim 1, which has a sequence from No. 190 to No. 190. 4. The DNA according to claim 1, wherein the transcription factor binding sequence comprises the nucleotide sequence shown in SEQ ID NO: 1. 5. The transcription factor binding sequence has a nucleotide sequence shown in SEQ ID NO: 1 in which one or several bases are deleted,
2. The DNA according to claim 1, comprising a substituted or added nucleotide sequence, and acting as a cis factor for increasing bacterial transcription. 6. A transcription factor binding sequence derived from an RNA polymerase III promoter-like sequence and comprising a transcription factor binding sequence that acts as a cis factor to increase bacterial transcription.
NA. 7. The RNA polymerase III promoter is a human genome repeat sequence Alu RNA polymerase.
The DNA according to claim 6, which is a III promoter. 8. The DNA according to claim 7, wherein the Alu sequence is a 3′-α1 Alu sequence located on the 3 ′ side of the human α1-globin gene. 9. The DNA according to claim 7, wherein the Alu sequence is a sequence containing a base sequence shown in the region from base position 1 to base position 88 of the 3′-α1 Alu fragment. 10. The transcription factor binding sequence comprises the nucleotide sequence shown in SEQ ID NO: 2.
DNA according to any one of the above. 11. A transcription factor binding sequence comprising a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 2, and a cis factor that increases bacterial transcription. The DNA according to claim 6, which acts as a DNA. 12. The DN according to any one of claims 1 to 11,
DN comprising a sequence complementary to sequence A
A. 13. A DNA, which is derived from Escherichia coli and is derived from a sequence having the same identity as the Escherichia coli F-element transposable region, which encodes a transcription factor that increases bacterial transcription activity. 14. The DN according to claim 13, which encodes the ArtA protein of Escherichia coli F factor.
A. 15. The DNA according to claim 13, which comprises the base sequence shown in SEQ ID NO: 3. 16. The method according to claim 13, wherein the base sequence shown in SEQ ID NO: 3 comprises a base sequence in which one or several bases have been deleted, substituted or added, and acts as a transcription factor. The described DNA. 17. D according to any one of claims 13 to 16,
DN comprising a sequence complementary to the NA sequence
A. 18. The D according to claim 1, wherein
A transcription factor comprising a protein that binds to NA and has an activity of increasing bacterial transcription. 19. A transcription factor, wherein the protein comprises the amino acid sequence shown in SEQ ID NO: 4. 20. An amino acid sequence represented by SEQ ID NO: 4 in which one or several amino acids are composed of an amino acid sequence in which deletion, substitution, or addition is performed, and a protein having an activity of increasing bacterial transcription. Characteristic transcription factor. 21. A transcription factor comprising a protein encoded by the DNA according to claim 13 and having a bacterial transcription activity. 22. D according to any one of claims 1 to 11,
NA and / or D according to any one of claims 13 to 16.
A bacterial expression vector, wherein NA is introduced into the vector. 23. The bacterial expression vector according to claim 22, wherein the vector has a promoter derived from T5 phage. 24. The bacterial expression vector according to claim 22, wherein the vector has a DNA encoding a predetermined protein. A bacterial transformant obtained by introducing the bacterial expression vector according to any one of claims 22 to 24 into a bacterial host. 26. The bacterial transformant according to claim 25, wherein the bacterial host is Escherichia coli. 27. A method for producing a protein, comprising culturing the transformant according to claim 25 or 26 to produce the protein.