CA2265884A1 - Rna polymerase - Google Patents
Rna polymeraseInfo
- Publication number
- CA2265884A1 CA2265884A1 CA002265884A CA2265884A CA2265884A1 CA 2265884 A1 CA2265884 A1 CA 2265884A1 CA 002265884 A CA002265884 A CA 002265884A CA 2265884 A CA2265884 A CA 2265884A CA 2265884 A1 CA2265884 A1 CA 2265884A1
- Authority
- CA
- Canada
- Prior art keywords
- rna polymerase
- amino acid
- wild type
- phage
- tyrosine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 title claims abstract 51
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 title claims abstract 51
- 150000001413 amino acids Chemical class 0.000 claims abstract 17
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims abstract 10
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims abstract 6
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims abstract 6
- 239000005547 deoxyribonucleotide Substances 0.000 claims abstract 5
- 239000002773 nucleotide Substances 0.000 claims abstract 5
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims abstract 5
- 125000000539 amino acid group Chemical group 0.000 claims 15
- 101710137500 T7 RNA polymerase Proteins 0.000 claims 9
- 230000037430 deletion Effects 0.000 claims 6
- 238000012217 deletion Methods 0.000 claims 6
- 230000037431 insertion Effects 0.000 claims 6
- 238000003780 insertion Methods 0.000 claims 6
- 238000006467 substitution reaction Methods 0.000 claims 6
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims 5
- 125000003729 nucleotide group Chemical group 0.000 claims 4
- 102000039446 nucleic acids Human genes 0.000 claims 3
- 108020004707 nucleic acids Proteins 0.000 claims 3
- 150000007523 nucleic acids Chemical class 0.000 claims 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims 2
- 238000012986 modification Methods 0.000 claims 2
- 230000004048 modification Effects 0.000 claims 2
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000035772 mutation Effects 0.000 claims 1
- 239000002157 polynucleotide Substances 0.000 claims 1
- 102000040430 polynucleotide Human genes 0.000 claims 1
- 108091033319 polynucleotide Proteins 0.000 claims 1
- 108091028664 Ribonucleotide Proteins 0.000 abstract 2
- 125000002637 deoxyribonucleotide group Chemical group 0.000 abstract 2
- 239000002336 ribonucleotide Substances 0.000 abstract 2
- 125000002652 ribonucleotide group Chemical group 0.000 abstract 2
- 238000010348 incorporation Methods 0.000 abstract 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
Abstract
An RNA polymerase comprising a wild type RNA polymerase with at least one amino acid modified so as to have a higher ability of incorporating 3'deoxyribonucleotide or a derivative thereof than that of the corresponding wild type RNA polymerase. Specifically, for example, an RNA polymerase comprising a wild type RNA polymerase wherein at least one amino acid present in the nucleotide bonding site, for example, phenylalanine, is substituted by tyrosine. The RNA polymerase has little or no bias of incorporation between ribonucleotide and 3'-deoxyribonucleotide, between ribonucleotides having different bases, and between deoxyribonucleotides having different bases.
Claims (25)
1. An RNA polymerase consisting of a wild type RNA
polymerase at least one of amino acids in the wild type RNA
polymerase is modified to enhance its ability for incorporating 3'-deoxyribonucleotides and derivatives thereof in comparison with the corresponding wild type RNA polymerase.
polymerase at least one of amino acids in the wild type RNA
polymerase is modified to enhance its ability for incorporating 3'-deoxyribonucleotides and derivatives thereof in comparison with the corresponding wild type RNA polymerase.
2. The RNA polymerase of claim 1, wherein at least one amino acid present in a nucleotide binding site of the wild type RNA
polymerase has been modified.
polymerase has been modified.
3. The RNA polymerase of claim 2, wherein the modification of amino acid is substitution, insertion or deletion of amino acid.
4. The RNA polymerase of any one of claims 1-3, wherein at least one amino acid present in the nucleotide binding site of the wild type RNA polymerase is replaced with tyrosine.
5. The RNA polymerase of claim 4, wherein the replaced amino acid is phenylalanine.
6. The RNA polymerase of any one of claims 2-5, wherein the amino acid present in the nucleotide binding site is an amino acid in a loop between helix Y and helix Z and/or an amino acid in a loop between helix Z and helix AA.
7. The RNA polymerase of any one of claims 1-6, which has been modified so that the ability for incorporating 3'-deoxyribonucleotides and derivatives thereof should be increased by twice in comparison with the wild type.
8. The RNA polymerase of any one of claims 1-7, which is derived from T7 phage, T3 phage, SP6 phage, or K11 phage.
9. An RNA polymerase consisting of a wild type RNA
polymerase provided that at least one of amino acids present in a region of the wild type RNA polymerase corresponding to amino acid residues 641-667 of RNA polymerase derived from T7 phage has been modified.
polymerase provided that at least one of amino acids present in a region of the wild type RNA polymerase corresponding to amino acid residues 641-667 of RNA polymerase derived from T7 phage has been modified.
10. The RNA polymerase of any one of claims 1-9, wherein the modified wild type RNA polymerase has further substitution, insertion or deletion of amino acid other than the modification.
11. An RNA polymerase which is an RNA polymerase derived from T7 phage, and has tyrosine at amino acid residue 644 or 667.
12. The RNA polymerase of claim 11, wherein the RNA
polymerase derived from T7 phage has further substitution, insertion, or deletion of amino acid other than the amino acid residues 644 and 667.
polymerase derived from T7 phage has further substitution, insertion, or deletion of amino acid other than the amino acid residues 644 and 667.
13. An RNA polymerase consisting of a wild type T7 RNA
polymerase provided that 644th amino acid residue of the wild type T7 RNA polymerase, phenylalanine, has been replaced with tyrosine.
polymerase provided that 644th amino acid residue of the wild type T7 RNA polymerase, phenylalanine, has been replaced with tyrosine.
14. An RNA polymerase consisting of a wild type T7 RNA
polymerase provided that 667th amino acid residue, phenylalanine, of the wild type T7 RNA polymerase has been replaced with tyrosine.
polymerase provided that 667th amino acid residue, phenylalanine, of the wild type T7 RNA polymerase has been replaced with tyrosine.
15. The RNA polymerase of claim 13 or 14, wherein 665th amino acid residue, leucine, of the wild type T7 RNA polymerase has been replaced with proline.
16. An RNA polymerase consisting of a wild type T7 RNA
polymerase provided that 644th amino acid residue, phenylalanine, of the wild type T7 RNA polymerase has been replaced with tyrosine, and 667th amino acid residue, phenylalanine, of the wild type T7 RNA polymerase has been replaced with tyrosine.
polymerase provided that 644th amino acid residue, phenylalanine, of the wild type T7 RNA polymerase has been replaced with tyrosine, and 667th amino acid residue, phenylalanine, of the wild type T7 RNA polymerase has been replaced with tyrosine.
17. The RNA polymerase of claim 16, wherein 665th amino acid residue, leucine, of the wild type T7 RNA polymerase has been replaced with proline.
18. An RNA polymerase which is an RNA polymerase derived from T3 phage, and has tyrosine at amino acid residue 645 or 668.
19. The RNA polymerase of claim 18, wherein the RNA
polymerase derived from T3 phage has further substitution, insertion, or deletion of amino acid other than the amino acid residues 645 and 668.
polymerase derived from T3 phage has further substitution, insertion, or deletion of amino acid other than the amino acid residues 645 and 668.
20. An RNA polymerase which is an RNA polymerase derived from K11 phage, and has tyrosine at one or more amino acid residues 664-669 and 690.
21. The RNA polymerase of claim 20, wherein the RNA
polymerase derived from K11 phage has further substitution, insertion, or deletion of amino acid other than the amino acid residues 664-669 and 690.
polymerase derived from K11 phage has further substitution, insertion, or deletion of amino acid other than the amino acid residues 664-669 and 690.
22. An RNA polymerase which is RNA polymerase derived from SP6 phage, and has tyrosine at one or more amino acid residues 633-638 and 670.
23. The RNA polymerase of claim 22, wherein the RNA
polymerase derived from SP6 phage has further substitution, insertion, or deletion of amino acid other than the amino acid residues 633-638 and 670.
polymerase derived from SP6 phage has further substitution, insertion, or deletion of amino acid other than the amino acid residues 633-638 and 670.
24. A polynucleotide encoding at least a part of RNA
polymerase of any one of claims 1-18.
polymerase of any one of claims 1-18.
25. A method for producing the RNA polymerase of any one of claims 1-23, which comprises:
preparing a nucleic acid molecule encoding an RNA polymerase, introducing a mutation into the nucleic acid molecule so that one or more nucleotides in one or more regions should be changed, and collecting a modified RNA polymerase expressed by the mutated nucleic acid molecule.
preparing a nucleic acid molecule encoding an RNA polymerase, introducing a mutation into the nucleic acid molecule so that one or more nucleotides in one or more regions should be changed, and collecting a modified RNA polymerase expressed by the mutated nucleic acid molecule.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9/180883 | 1997-07-07 | ||
| JP18088397 | 1997-07-07 | ||
| JP15575998A JP3172710B2 (en) | 1997-07-07 | 1998-06-04 | RNA polymerase |
| JP10/155759 | 1998-06-04 | ||
| PCT/JP1998/003037 WO1999002698A1 (en) | 1997-07-07 | 1998-07-06 | Rna polymerase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2265884A1 true CA2265884A1 (en) | 1999-01-21 |
| CA2265884C CA2265884C (en) | 2010-06-22 |
Family
ID=26483680
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2265884A Expired - Fee Related CA2265884C (en) | 1997-07-07 | 1998-07-06 | Rna polymerase |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US6867027B1 (en) |
| EP (1) | EP0939130B1 (en) |
| JP (1) | JP3172710B2 (en) |
| CA (1) | CA2265884C (en) |
| DE (1) | DE69833112D1 (en) |
| WO (1) | WO1999002698A1 (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2301495T3 (en) * | 1999-11-05 | 2008-07-01 | Jena Bioscience Gmbh | PROTEIN EXPRESSION SYSTEMS FOR KINETOPLASTIDAE NOT PATHOGEN. |
| DE60110564T2 (en) * | 2000-03-07 | 2006-01-19 | bioMérieux B.V. | RNA POLYMERASE MUTANTS WITH INCREASED THERMOSTABILITY |
| FR2823219B1 (en) | 2001-04-10 | 2003-07-04 | Pasteur Institut | MUTANTS OF DESOXYCYTIDINE KINASE WITH ENLARGED ENZYMATIC ACTIVITY |
| JP2002360256A (en) * | 2001-06-04 | 2002-12-17 | Inst Of Physical & Chemical Res | Method for producing rna probe, method for detecting target nucleic acid, and kit for producing rna probe |
| JP2003061684A (en) * | 2001-06-11 | 2003-03-04 | Nippon Genetech Co Ltd | Method for dna base sequence determination |
| JP2003061683A (en) * | 2001-06-11 | 2003-03-04 | Nippon Genetech Co Ltd | Variation rna polymerase |
| US8101385B2 (en) | 2005-06-30 | 2012-01-24 | Archemix Corp. | Materials and methods for the generation of transcripts comprising modified nucleotides |
| CA2613442C (en) | 2005-06-30 | 2016-08-23 | Archemix Corp. | Materials and methods for the generation of fully 2'-modified nucleic acid transcripts |
| JP2009136153A (en) * | 2007-12-03 | 2009-06-25 | Tosoh Corp | Variant rna polymerase |
| JP5625908B2 (en) * | 2008-08-08 | 2014-11-19 | 東ソー株式会社 | RNA polymerase variants with improved function |
| CA3066767A1 (en) | 2017-06-30 | 2019-01-03 | Codexis, Inc. | T7 rna polymerase variants |
| KR20200023455A (en) | 2017-06-30 | 2020-03-04 | 코덱시스, 인코포레이티드 | T7 RNA Polymerase Variants |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5385834A (en) | 1993-08-13 | 1995-01-31 | Georgia Tech Research Corporation | Mutant T7 RNA polymerase GP1(lys222) exhibiting altered promoter recognition |
| US5614365A (en) * | 1994-10-17 | 1997-03-25 | President & Fellow Of Harvard College | DNA polymerase having modified nucleotide binding site for DNA sequencing |
| EP0655506B1 (en) | 1994-10-17 | 1996-09-18 | President And Fellows Of Harvard College | DNA polymerases having modified nucleotide binding site for DNA sequencing |
| EP0785278B1 (en) | 1994-11-07 | 2003-02-05 | The Institute Of Physical & Chemical Research | Method for determining dna nucleotide sequence |
| JP3318579B2 (en) * | 1997-07-07 | 2002-08-26 | 理化学研究所 | DNA sequencing method |
-
1998
- 1998-06-04 JP JP15575998A patent/JP3172710B2/en not_active Expired - Lifetime
- 1998-07-06 EP EP98929851A patent/EP0939130B1/en not_active Expired - Lifetime
- 1998-07-06 US US09/254,344 patent/US6867027B1/en not_active Expired - Fee Related
- 1998-07-06 CA CA2265884A patent/CA2265884C/en not_active Expired - Fee Related
- 1998-07-06 DE DE69833112T patent/DE69833112D1/en not_active Expired - Lifetime
- 1998-07-06 WO PCT/JP1998/003037 patent/WO1999002698A1/en active IP Right Grant
Also Published As
| Publication number | Publication date |
|---|---|
| EP0939130A1 (en) | 1999-09-01 |
| US6867027B1 (en) | 2005-03-15 |
| EP0939130B1 (en) | 2006-01-04 |
| WO1999002698A1 (en) | 1999-01-21 |
| JP3172710B2 (en) | 2001-06-04 |
| EP0939130A4 (en) | 2002-09-18 |
| CA2265884C (en) | 2010-06-22 |
| DE69833112D1 (en) | 2006-03-30 |
| JPH1175867A (en) | 1999-03-23 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |