Claims
- 1. A method to identify one or more agents which inhibit or prevent the binding of a moiety to the flap-tip helix of the β subunit of core RNA polymerase, comprising:
a) providing a complex formed by contacting the one or more agents with isolated core RNA polymerase, or an isolated β subunit of RNA polymerase or a portion thereof which comprises the flap-tip helix; b) contacting the complex with a moiety which specifically binds the flap-tip helix; and C) detecting or determining whether the one or more agents inhibit or prevent the binding of the moiety to the flap-tip helix.
- 2. A method to identify one or more agents which inhibit or prevent the binding of a moiety to the flap-tip helix of the β subunit of core RNA polymerase, comprising:
a) providing a complex formed by contacting the one or more agents with a moiety which specifically binds the flap-tip helix; b) contacting the complex with isolated core RNA polymerase, or an isolated β subunit of RNA polymerase or a portion thereof which comprises the flap-tip helix; and c) detecting or determining whether the one or more agents inhibit or prevent the binding of the moiety to the flap-tip helix.
- 3. A method to identify one or more moieties which bind to the flap-tip helix, comprising:
a) contacting a peptide comprising the flap-tip helix or a portion thereof with the one or more moieties; and b) detecting or determining whether the one or more moieties specifically bind to the flap-tip helix.
- 4. The method of claim 1, 2 or 3 wherein the moiety is a nucleic acid aptamer.
- 5. The method of claim 1, 2 or 3 wherein the moiety is a peptide aptamer.
- 6. The method of claim 1, 2 or 3 wherein the moiety is a prokaryotic protein.
- 7. The method of claim 6 wherein the protein is a transcription factor.
- 8. The method of claim 7 wherein the transcription factor is an initiation factor.
- 9. The method of claim 7 wherein the transcription factor is an elongation factor.
- 10. The method of claim 8 wherein the initiation factor is σ.
- 11. The method of claim 10 wherein the initiation factor is σ70.
- 12. The method of claim 9 wherein the elongation factor is NusA.
- 13. The method of claim 1, 2 or 3 wherein the portion includes residues corresponding to residues 900 to 909 of the β subunit of E. coli RNA polymerase.
- 14. The method of claim 1, 2 or 3 wherein the portion includes residues corresponding to residues 897 to 907 of the β subunit of E. coli RNA polymerase.
- 15. The method of claim 1, 2 or 3 wherein the portion includes residues corresponding to residues 830 to 1058 of the β subunit of E. coli RNA polymerase.
- 16. The method of claim 1 or 2 wherein core RNA polymerase, the β subunit or a portion thereof is labeled or is capable of being bound by a detectable label.
- 17. The method of claim 16 wherein a fluorophore is attached to or near the flap-tip helix.
- 18. The method of claim 16 wherein core RNA polymerase, the β subunit or portion thereof is capable of being bound by a labeled antibody.
- 19. The method of claim 1, 2 or 3 wherein the moiety is labeled or is capable of being bound by a detectable label.
- 20. The method of claim 19 wherein the moiety is capable of being bound by a labeled antibody.
- 21. The method of claim 3 wherein the peptide is labeled or is capable of being bound by a detectable label.
- 22. The method of claim 1 or 2 wherein fluorescence resonance energy transfer is employed to detect or determine whether the agent inhibits or prevents binding.
- 23. The method of claim 1 or 2 wherein luminescence resonance energy transfer is employed to detect or determine whether the agent inhibits or prevents binding.
- 24. The method of claim 1 or 2 wherein trypsin cleavage and SDS-PAGE are employed to detect or determine whether the agent inhibits or prevents binding.
- 25. The method of claim 1 wherein core RNA polymerase or the β subunit or portion thereof is attached to a solid substrate.
- 26. The method of claim 25 wherein the solid support comprises a scintillant and the moiety is radioactively labeled.
- 27. The method of claim 2 wherein the moiety is attached to a solid substrate.
- 28. The method of claim 27 wherein the solid support comprises a scintillant and core RNA polymerase, β subunit or portion thereof is radioactively labeled.
- 29. The method of claim 3 wherein the peptide is attached to a solid support.
- 30. The method of claim 29 wherein the solid support comprises a scintillant and the moiety is radioactively labeled.
- 31. The method of claim 3 wherein the one or more moieties are products encoded by a recombinant phage, virus, or cell.
- 32. The method of claim 1, 2 or 3 wherein a protease or nuclease is attached to or near the flap-tip helix.
- 33. The method of claim 1, 2 or 3 wherein a cross-linking moiety is attached to or near the flap-tip helix.
- 34. An agent identified by the method of claim 1 or 2.
- 35. A method to inhibit the growth of a prokaryotic cell, comprising:
contacting the cell with an effective amount of the agent of claim 34.
- 36. An isolated and purified portion of the β subunit of RNA polymerase which binds to NusA or σ.
- 37. The isolated and purified portion of the β subunit of claim 36 which comprises a portion selected from the group consisting of residues corresponding to residues 830 to 1058, residues 897 to 907, and residues 900 to 909 of the β subunit of E. coli RNA polymerase.
- 38. A method to identify one or more agents that which inhibit or prevent the binding of a prokaryotic protein to the flap-tip helix of the β subunit of core RNA polymerase, comprising:
a) contacting the one or more agents with a recombinant cell which expresses a first fusion polypeptide and a second fusion polypeptide, wherein the first fusion polypeptide comprises the flap-tip helix and a first polypeptide and the second fusion polypeptide comprises the prokaryotic protein or a fragment thereof which specifically binds the flap-tip helix and a second polypeptide, wherein the binding of the first polypeptide to the second polypeptide yields a detectable signal; and b) detecting or determining whether the one or more agents inhibits or prevents the signal.
- 39. The method of claim 38 wherein either the first or the second polypeptide comprises a DNA binding domain.
- 40. The method of claim 38 wherein either the first or the second polypeptide comprises an activation domain.
- 41. A moiety identified by the method of claim 3.
- 42. A method to identify one or more agents which inhibit or prevent transcription from a promoter that is dependent on a −35 sequence for activity, comprising:
a) contacting the one or more agents with a composition for transcription comprising wild-type RNA polymerase and a first construct comprising a first promoter that is dependent on a −35 sequence to promote transcription and operably linked to an open reading frame for a gene; and b) identifying an agent that inhibits or prevents transcription from the first construct relative to transcription by wild-type RNA polymerase from a second construct comprising a second promoter that promotes transcription independent of the presence of a −35 sequence, which second promoter is operably linked to an open reading frame for a gene, thereby identifying an agent that inhibits or prevents transcription from a promoter that is dependent on a −35 sequence for activity.
- 43. The method of claim 42 wherein the one or more agents are contacted with a recombinant cell augmented with the first construct.
- 44. The method of claim 42 wherein the level of transcription from the first construct in the presence of the agent is substantially the same as the level of transcription by a mutant RNA polymerase comprising a mutant flap-tip helix from a third construct comprising a promoter that is dependent on a −35 sequence to promote transcription and operably linked to an open reading frame for a gene.
- 45. The method of claim 42 wherein the agent does not inhibit transcription by a mutant RNA polymerase comprising a mutant flap-tip helix from a third construct comprising a promoter that promotes transcription independent of the presence of a −35 sequence and operably linked to an open reading frame for a gene.
- 46. The method of claim 42 wherein the open reading frame in the first construct is an open reading frame for a marker gene or a selectable gene.
- 47. The method of claim 42 wherein the open reading frame in the second construct is an open reading frame for a marker gene or a selectable gene.
- 48. The method of claim 42 wherein the open reading frame in the first construct is different that the open reading frame in the second construct.
- 49. The method of claim 42 wherein the recombinant cell further comprises the second construct and wherein the open reading frame in the first construct is different that the open reading frame in the second construct.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of U.S. application Ser. No. 60/286,783 filed on Apr. 25, 2001, under 35 U.S.C. § 119(e), the disclosure of which is incorporated by reference herein.
STATEMENT OF GOVERNMENT RIGHTS
[0002] This invention was made at least in part with a grant from the Government of the United States (grant GM 38660 from the National Institutes of Health). The Government has certain rights to the invention.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60286783 |
Apr 2001 |
US |