Recombinant Thermus aquaticus RNA Polymerase for Structural Studies
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, labo...
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creator | Kuznedelov, Konstantin Lamour, Valerie Patikoglou, Georgia Chlenov, Mark Darst, Seth A. Severinov, Konstantin |
description | 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 β′ 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
Taqβ′NCD deletion mutants generated in this study showed that the β′NCD is important for the efficient binding of the σ subunit, confirming previous hypotheses. Finally, preliminary structural analysis (at 4.1
Å resolution) of one of the recombinant mutants revealed a previously unobserved conformation of the β-flap, further defining the range of conformations accessible to this flexible structural element. |
doi_str_mv | 10.1016/j.jmb.2006.03.009 |
format | Article |
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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 β′ 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
Taqβ′NCD deletion mutants generated in this study showed that the β′NCD is important for the efficient binding of the σ subunit, confirming previous hypotheses. Finally, preliminary structural analysis (at 4.1
Å resolution) of one of the recombinant mutants revealed a previously unobserved conformation of the β-flap, further defining the range of conformations accessible to this flexible structural element.</description><identifier>ISSN: 0022-2836</identifier><identifier>EISSN: 1089-8638</identifier><identifier>DOI: 10.1016/j.jmb.2006.03.009</identifier><identifier>PMID: 16618493</identifier><language>eng</language><publisher>England: Elsevier Ltd</publisher><subject>BACTERIA ; Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; BASIC BIOLOGICAL SCIENCES ; BIOLOGY ; crystal structure ; Crystallization - methods ; Crystallography, X-Ray ; DNA-Directed RNA Polymerases - chemistry ; DNA-Directed RNA Polymerases - genetics ; DNA-Directed RNA Polymerases - metabolism ; ENZYMES ; ESCHERICHIA COLI ; Eubacteria ; Eubacterium ; Evolution, Molecular ; GENETICS ; Models, Molecular ; Molecular Sequence Data ; MUTANTS ; national synchrotron light source ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; RESOLUTION ; RNA polymerase ; RNA POLYMERASES ; Thermus - enzymology ; Thermus aquaticus ; Thermus thermophilus ; TRANSCRIPTION</subject><ispartof>Journal of molecular biology, 2006-05, Vol.359 (1), p.110-121</ispartof><rights>2006 Elsevier Ltd</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c408t-53e6a39598da3e5bdc55eca5db7b00999c9b3580db5acc4c5910d98bea1770ab3</citedby><cites>FETCH-LOGICAL-c408t-53e6a39598da3e5bdc55eca5db7b00999c9b3580db5acc4c5910d98bea1770ab3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jmb.2006.03.009$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>230,314,780,784,885,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16618493$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://www.osti.gov/biblio/914274$$D View this record in Osti.gov$$Hfree_for_read</backlink></links><search><creatorcontrib>Kuznedelov, Konstantin</creatorcontrib><creatorcontrib>Lamour, Valerie</creatorcontrib><creatorcontrib>Patikoglou, Georgia</creatorcontrib><creatorcontrib>Chlenov, Mark</creatorcontrib><creatorcontrib>Darst, Seth A.</creatorcontrib><creatorcontrib>Severinov, Konstantin</creatorcontrib><creatorcontrib>Brookhaven National Laboratory (BNL) National Synchrotron Light Source</creatorcontrib><title>Recombinant Thermus aquaticus RNA Polymerase for Structural Studies</title><title>Journal of molecular biology</title><addtitle>J Mol Biol</addtitle><description>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 β′ 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
Taqβ′NCD deletion mutants generated in this study showed that the β′NCD is important for the efficient binding of the σ subunit, confirming previous hypotheses. Finally, preliminary structural analysis (at 4.1
Å resolution) of one of the recombinant mutants revealed a previously unobserved conformation of the β-flap, further defining the range of conformations accessible to this flexible structural element.</description><subject>BACTERIA</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>BASIC BIOLOGICAL SCIENCES</subject><subject>BIOLOGY</subject><subject>crystal structure</subject><subject>Crystallization - methods</subject><subject>Crystallography, X-Ray</subject><subject>DNA-Directed RNA Polymerases - chemistry</subject><subject>DNA-Directed RNA Polymerases - genetics</subject><subject>DNA-Directed RNA Polymerases - metabolism</subject><subject>ENZYMES</subject><subject>ESCHERICHIA COLI</subject><subject>Eubacteria</subject><subject>Eubacterium</subject><subject>Evolution, Molecular</subject><subject>GENETICS</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>MUTANTS</subject><subject>national synchrotron light source</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>RESOLUTION</subject><subject>RNA polymerase</subject><subject>RNA POLYMERASES</subject><subject>Thermus - enzymology</subject><subject>Thermus aquaticus</subject><subject>Thermus thermophilus</subject><subject>TRANSCRIPTION</subject><issn>0022-2836</issn><issn>1089-8638</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUtr3DAUhUVIaaZJf0A2xdl0Z_fKsmyJrMLQF4Q05LEWetwhGmwrkeRA_n01zEB36erexXcP95xDyDmFhgLtv22b7WSaFqBvgDUA8oisKAhZi56JY7ICaNu6Faw_IZ9S2gIAZ534SE5o31PRSbYi6zu0YTJ-1nOuHp4wTkuq9Muis7dlu7u5qm7D-DZh1AmrTYjVfY6LzUvUY1kX5zGdkQ8bPSb8fJin5PHH94f1r_r6z8_f66vr2nYgcs0Z9ppJLoXTDLlxlnO0mjszmPK7lFYaxgU4w7W1neWSgpPCoKbDANqwU3Kx1w0pe5Wsz2ifbJhntFlJ2rVDV5ive-Y5hpcFU1aTTxbHUc8YlqT6QXZtSeG_IB2ooG0rC0j3oI0hpYgb9Rz9pOOboqB2NaitKjWoXQ0KmCpWys2Xg_hiJnT_Lg65F-ByD2DJ69Vj3NnB2aLzcefGBf-O_F-I7Jf0</recordid><startdate>20060526</startdate><enddate>20060526</enddate><creator>Kuznedelov, Konstantin</creator><creator>Lamour, Valerie</creator><creator>Patikoglou, Georgia</creator><creator>Chlenov, Mark</creator><creator>Darst, Seth A.</creator><creator>Severinov, Konstantin</creator><general>Elsevier Ltd</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7TM</scope><scope>C1K</scope><scope>7X8</scope><scope>OTOTI</scope></search><sort><creationdate>20060526</creationdate><title>Recombinant Thermus aquaticus RNA Polymerase for Structural Studies</title><author>Kuznedelov, Konstantin ; Lamour, Valerie ; Patikoglou, Georgia ; Chlenov, Mark ; Darst, Seth A. ; Severinov, Konstantin</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c408t-53e6a39598da3e5bdc55eca5db7b00999c9b3580db5acc4c5910d98bea1770ab3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>BACTERIA</topic><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>BASIC BIOLOGICAL SCIENCES</topic><topic>BIOLOGY</topic><topic>crystal structure</topic><topic>Crystallization - methods</topic><topic>Crystallography, X-Ray</topic><topic>DNA-Directed RNA Polymerases - chemistry</topic><topic>DNA-Directed RNA Polymerases - genetics</topic><topic>DNA-Directed RNA Polymerases - metabolism</topic><topic>ENZYMES</topic><topic>ESCHERICHIA COLI</topic><topic>Eubacteria</topic><topic>Eubacterium</topic><topic>Evolution, Molecular</topic><topic>GENETICS</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>MUTANTS</topic><topic>national synchrotron light source</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>RESOLUTION</topic><topic>RNA polymerase</topic><topic>RNA POLYMERASES</topic><topic>Thermus - enzymology</topic><topic>Thermus aquaticus</topic><topic>Thermus thermophilus</topic><topic>TRANSCRIPTION</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kuznedelov, Konstantin</creatorcontrib><creatorcontrib>Lamour, Valerie</creatorcontrib><creatorcontrib>Patikoglou, Georgia</creatorcontrib><creatorcontrib>Chlenov, Mark</creatorcontrib><creatorcontrib>Darst, Seth A.</creatorcontrib><creatorcontrib>Severinov, Konstantin</creatorcontrib><creatorcontrib>Brookhaven National Laboratory (BNL) National Synchrotron Light Source</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Nucleic Acids Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><collection>OSTI.GOV</collection><jtitle>Journal of molecular biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kuznedelov, Konstantin</au><au>Lamour, Valerie</au><au>Patikoglou, Georgia</au><au>Chlenov, Mark</au><au>Darst, Seth A.</au><au>Severinov, Konstantin</au><aucorp>Brookhaven National Laboratory (BNL) National Synchrotron Light Source</aucorp><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Recombinant Thermus aquaticus RNA Polymerase for Structural Studies</atitle><jtitle>Journal of molecular biology</jtitle><addtitle>J Mol Biol</addtitle><date>2006-05-26</date><risdate>2006</risdate><volume>359</volume><issue>1</issue><spage>110</spage><epage>121</epage><pages>110-121</pages><issn>0022-2836</issn><eissn>1089-8638</eissn><abstract>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 β′ 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
Taqβ′NCD deletion mutants generated in this study showed that the β′NCD is important for the efficient binding of the σ subunit, confirming previous hypotheses. Finally, preliminary structural analysis (at 4.1
Å resolution) of one of the recombinant mutants revealed a previously unobserved conformation of the β-flap, further defining the range of conformations accessible to this flexible structural element.</abstract><cop>England</cop><pub>Elsevier Ltd</pub><pmid>16618493</pmid><doi>10.1016/j.jmb.2006.03.009</doi><tpages>12</tpages></addata></record> |
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subjects | BACTERIA Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - metabolism BASIC BIOLOGICAL SCIENCES BIOLOGY crystal structure Crystallization - methods Crystallography, X-Ray DNA-Directed RNA Polymerases - chemistry DNA-Directed RNA Polymerases - genetics DNA-Directed RNA Polymerases - metabolism ENZYMES ESCHERICHIA COLI Eubacteria Eubacterium Evolution, Molecular GENETICS Models, Molecular Molecular Sequence Data MUTANTS national synchrotron light source Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism RESOLUTION RNA polymerase RNA POLYMERASES Thermus - enzymology Thermus aquaticus Thermus thermophilus TRANSCRIPTION |
title | Recombinant Thermus aquaticus RNA Polymerase for Structural Studies |
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