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Bell, Stephen D.; Jackson, Stephen

doi:10.1038/78907

Cited by 140 publications

(39 citation statements)

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“…The major fluorophore monitored for IPTG binding is W220, which exhibits a shift of the spectrum to lower wavelength upon LacI binding to inducer ( − ), a situation presented even in V52W. All LacI variants exhibit IPTG binding affinity comparable to that of the wild-type protein (Table ), consistent with the anticipation that the conformation of the inducer-binding pocket is unchanged in these V52 mutants ( − ).…”

Section: Resultsmentioning

confidence: 66%

“…This 150 kDa homotetramer is a dimer of functionally independent dimers, with one operator binding site and two sugar-binding sites per dimer (Figure A). Each monomer consists of distinct domains: a helix−turn−helix (HTH) N-terminal DNA-binding domain; a hinge-helix region; a core domain (including N- and C-subdomains), with the inducer-binding site sandwiched between the subdomains; and a C-terminal tetramerization domain ( − ). The hinge helix is the covalent connection between the two functional domains.…”

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confidence: 99%

“…Asn50 and Asn51 contact the N-subdomain of the partner monomer, and Gln55 interacts with the core N-subdomain of the same monomer (Figure B). All of these interfaces are interrupted by the conformational change triggered by IPTG binding to the core domain ( , ).…”

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confidence: 99%

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Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

2006

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A significant number of eukaryotic regulatory proteins are predicted to have disordered regions. Many of these proteins bind DNA, which may serve as a template for protein folding. Similar behavior is seen in the prokaryotic LacI/GalR family of proteins that couple hinge-helix folding with DNA binding. These hinge regions form short α-helices when bound to DNA, but appear to be disordered in other states. An intriguing question is whether and to what degree intrinsic helix propensity contributes to the function of these proteins. In addition to its interaction with operator DNA, the LacI hinge helix interacts with the hinge helix of the homodimer partner as well as to the surface of the inducer-binding domain. To explore the hierarchy of these interactions, we made a series of substitutions in the LacI hinge helix at position 52, the only site in the helix that does not interact with DNA and/or the inducer-binding domain. The substitutions at V52 have significant effects on operator binding affinity and specificity, and several substitutions also impair functional communication with the inducer-binding domain. Results suggest that helical propensity of amino acids in the hinge region alone does not dominate function; helix-helix packing interactions appear to also contribute. Further, the data demonstrate that variation in operator sequence can overcome side chain effects on hinge helix folding and/or hinge•hinge interactions. Thus, this system provides a direct example whereby an extrinsic interaction (DNA binding) guides internal events that influence folding and functionality. KeywordsLacI; helical propensity; allostery; DNA binding Protein folding has long been a fundamental issue in biological sciences. Beyond the general question of how a polypeptide sequence adopts a three-dimensional structure, coupled folding and binding have been identified as a key feature of partially unstructured proteins in multiple regulatory processes (e.g., transcription regulation, signal transduction, membrane transport and signaling (6-9)). Indeed, more and more intrinsically disordered proteins are found to fold upon binding to their target partners, ligands, or even small ions (10-13). In the case of transcription regulation, mutual cooperative folding of both protein and DNA has been † This work was supported by NIH Grant GM22441 and Robert A. Welch Grant C-576 to Kathleen Shive Matthews. Liskin Swint-Kruse is supported by NIH Grant P20 RR17708 from the Institutional Development Award program of the National Center for Research Resources.*To whom correspondence should be addressed. Telephone: 713−348−4871; Fax: 713−348−6149; Email: E-mail: ksm@bioc.rice.edu.. observed (6,11,14,15). In this paper, coupled folding is explored in greater detail for the hinge helix of the lactose repressor protein (LacI) 1 . NIH Public AccessLacI is a premier model for the regulation of gene expression and allosteric transition in biological systems (2) 2 . This 150-kDa homotetramer is a dimer of functionally independent dimers, with ...

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Section: Resultsmentioning

confidence: 66%

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confidence: 99%

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Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

2006

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“…Recent crystallographic structures of bacterial RNAP and eukaryal RNAP II reveal striking structural homology (3, 14 -20), homology that apparently also extends to archaeal RNAP (3,(21)(22)(23). Archaeal and eukaryal general transcription factors also exhibit striking structural homology (24 -29).…”

Section: Comparison With Crystallographic Information Implicates the mentioning

confidence: 99%

“…Transcription initiation in archaea closely resembles eukaryal class II transcription initiation and is mediated by a single RNA polymerase (RNAP) 1 and two general transcription factors, TATA-element binding protein (TBP) and transcription factor B (TFB) (5, 6). In vitro transcription initiation has been demonstrated with RNAP, TBP, and TFB in Pyrococcus (7-10), Methanococcus (11), Methanosarcina (12), and Sulfolobus (13) cell-free transcription systems.Recent crystallographic structures of bacterial RNAP and eukaryal RNAP II reveal striking structural homology (3, 14 -20), homology that apparently also extends to archaeal RNAP (3,(21)(22)(23). Archaeal and eukaryal general transcription factors also exhibit striking structural homology (24 -29).…”

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Transcription Factor B Contacts Promoter DNA Near the Transcription Start Site of the Archaeal Transcription Initiation Complex

Renfrow

Naryshkin

Lewis

et al. 2004

Journal of Biological Chemistry

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Transcription initiation in all three domains of life requires the assembly of large multiprotein complexes at DNA promoters before RNA polymerase (RNAP)-catalyzed transcript synthesis. Core RNAP subunits show homology among the three domains of life, and recent structural information supports this homology. General transcription factors are required for productive transcription initiation complex formation. The archaeal general transcription factors TATA-element-binding protein (TBP), which mediates promoter recognition, and transcription factor B (TFB), which mediates recruitment of RNAP, show extensive homology to eukaryal TBP and TFIIB. Crystallographic information is becoming available for fragments of transcription initiation complexes (e.g. RNAP, TBP-TFB-DNA, TBP-TFIIB-DNA), but understanding the molecular topography of complete initiation complexes still requires biochemical and biophysical characterization of protein-protein and protein-DNA interactions. In published work, systematic site-specific protein-DNA photocrosslinking has been used to define positions of RNAP subunits and general transcription factors in bacterial and eukaryal initiation complexes. In this work, we have used systematic site-specific protein-DNA photocrosslinking to define positions of RNAP subunits and general transcription factors in an archaeal initiation complex. Employing a set of 41 derivatized DNA fragments, each having a phenyl azide photoactivable crosslinking agent incorporated at a single, defined site within positions ؊40 to ؉1 of the gdh promoter of the hyperthermophilic marine archaea, Pyrococcus furiosus (Pf), we have determined the locations of PfRNAP subunits PfTBP and PfTFB relative to promoter DNA. The resulting topographical information supports the striking homology with the eukaryal initiation complex and permits one major new conclusion, which is that PfTFB interacts with promoter DNA not only in the TATA-element region but also in the transcription-bubble region, near the transcription start site. Comparison with crystallographic information implicates the PfTFB N-terminal domain in the interaction with the transcription-bubble region. The results are discussed in relation to the known effects of substitutions in the TFB and TFIIB N-terminal domains on transcription initiation and transcription start-site selection.Extensive structural and biochemical characterization of transcription initiation complexes has revealed similarity of transcription systems across the three domains of life (1-4). Transcription initiation in archaea closely resembles eukaryal class II transcription initiation and is mediated by a single RNA polymerase (RNAP) 1 and two general transcription factors, TATA-element binding protein (TBP) and transcription factor B (TFB) (5, 6). In vitro transcription initiation has been demonstrated with RNAP, TBP, and TFB in Pyrococcus (7-10), Methanococcus (11), Methanosarcina (12), and Sulfolobus (13) cell-free transcription systems.Recent crystallographic structures of bacterial RNAP and eukary...

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Transcription Factors

Kohler

Allemann

2002

Encyclopedia of Molecular Biology

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Untitled

Cited by 140 publications

References 12 publications

Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

Transcription Factor B Contacts Promoter DNA Near the Transcription Start Site of the Archaeal Transcription Initiation Complex

Transcription Factors

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