2gho: Difference between revisions
New page: left|200px<br /><applet load="2gho" size="450" color="white" frame="true" align="right" spinBox="true" caption="2gho, resolution 5.000Å" /> '''Recombinant Thermus... |
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[[Image:2gho.gif|left|200px]]<br /><applet load="2gho" size=" | [[Image:2gho.gif|left|200px]]<br /><applet load="2gho" size="350" color="white" frame="true" align="right" spinBox="true" | ||
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'''Recombinant Thermus aquaticus RNA polymerase for Structural Studies'''<br /> | '''Recombinant Thermus aquaticus RNA polymerase for Structural Studies'''<br /> | ||
==Overview== | ==Overview== | ||
Advances in the structural biology of bacterial transcription have come | Advances in the structural biology of bacterial transcription have come from studies of RNA polymerases (RNAPs) from the thermophilic eubacteria Thermus aquaticus (Taq) and Thermus thermophilus (Tth). These structural studies have been limited by the fact that only endogenous Taq or Tth RNAP, laboriously purified from large quantities of Taq or Tth cell paste and offering few options for genetic modification, is suitable for structural studies. Recombinant systems for the preparation of Taq RNAP by co-overexpression and assembly in the heterologous host, Escherichia coli, have been described, but these did not yield enzyme suitable for crystallographic studies. Here we describe recombinant systems for the preparation of Taq RNAP harboring full or partial deletions of the Taq beta' non-conserved domain (NCD), yielding enzyme suitable for crystallographic studies. This opens the way for structural studies of genetically manipulated enzymes, allowing the preparation of more crystallizable enzymes and facilitating detailed structure/function analysis. Characterization of the Taqbeta'NCD deletion mutants generated in this study showed that the beta'NCD is important for the efficient binding of the sigma subunit, confirming previous hypotheses. Finally, preliminary structural analysis (at 4.1Angstroms resolution) of one of the recombinant mutants revealed a previously unobserved conformation of the beta-flap, further defining the range of conformations accessible to this flexible structural element. | ||
==About this Structure== | ==About this Structure== | ||
2GHO is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Thermus_aquaticus Thermus aquaticus]. Active as [http://en.wikipedia.org/wiki/DNA-directed_RNA_polymerase DNA-directed RNA polymerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.6 2.7.7.6] Full crystallographic information is available from [http:// | 2GHO is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Thermus_aquaticus Thermus aquaticus]. Active as [http://en.wikipedia.org/wiki/DNA-directed_RNA_polymerase DNA-directed RNA polymerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.6 2.7.7.6] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2GHO OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Protein complex]] | [[Category: Protein complex]] | ||
[[Category: Thermus aquaticus]] | [[Category: Thermus aquaticus]] | ||
[[Category: Darst, S | [[Category: Darst, S A.]] | ||
[[Category: Lamour, V.]] | [[Category: Lamour, V.]] | ||
[[Category: coexpression]] | [[Category: coexpression]] | ||
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[[Category: thermus aquaticus rna polymerase]] | [[Category: thermus aquaticus rna polymerase]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:31:47 2008'' | ||
Revision as of 18:31, 21 February 2008
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Recombinant Thermus aquaticus RNA polymerase for Structural Studies
OverviewOverview
Advances in the structural biology of bacterial transcription have come from studies of RNA polymerases (RNAPs) from the thermophilic eubacteria Thermus aquaticus (Taq) and Thermus thermophilus (Tth). These structural studies have been limited by the fact that only endogenous Taq or Tth RNAP, laboriously purified from large quantities of Taq or Tth cell paste and offering few options for genetic modification, is suitable for structural studies. Recombinant systems for the preparation of Taq RNAP by co-overexpression and assembly in the heterologous host, Escherichia coli, have been described, but these did not yield enzyme suitable for crystallographic studies. Here we describe recombinant systems for the preparation of Taq RNAP harboring full or partial deletions of the Taq beta' non-conserved domain (NCD), yielding enzyme suitable for crystallographic studies. This opens the way for structural studies of genetically manipulated enzymes, allowing the preparation of more crystallizable enzymes and facilitating detailed structure/function analysis. Characterization of the Taqbeta'NCD deletion mutants generated in this study showed that the beta'NCD is important for the efficient binding of the sigma subunit, confirming previous hypotheses. Finally, preliminary structural analysis (at 4.1Angstroms resolution) of one of the recombinant mutants revealed a previously unobserved conformation of the beta-flap, further defining the range of conformations accessible to this flexible structural element.
About this StructureAbout this Structure
2GHO is a Protein complex structure of sequences from Thermus aquaticus. Active as DNA-directed RNA polymerase, with EC number 2.7.7.6 Full crystallographic information is available from OCA.
ReferenceReference
Recombinant Thermus aquaticus RNA polymerase for structural studies., Kuznedelov K, Lamour V, Patikoglou G, Chlenov M, Darst SA, Severinov K, J Mol Biol. 2006 May 26;359(1):110-21. Epub 2006 Mar 23. PMID:16618493
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