Isolation of the Lac Repressor

Gilbert, Walter; Müller‐Hill, Benno

doi:10.1073/pnas.56.6.1891

Cited by 614 publications

(254 citation statements)

References 11 publications

Supporting

Mentioning

236

Contrasting

Unclassified

Order By: Relevance

“…The program Igor Pro (Wavemetrics, CA) was used to fit data for affinity assays using the equation: (1) where Y obs is the radioactivity retained at a specific protein concentration, Y max is the measured radioactivity when all of the DNA is bound to repressor, K d is the equilibrium dissociation constant, and c is the background radioactivity in the absence of protein. The value of the Hill coefficient, n, was either fixed at 1 or allowed to float, in which case the values are found to be ∼1.…”

Section: Dna Binding/operator Releasementioning

confidence: 99%

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

2006

View full text Add to dashboard Cite

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 ...

show abstract

Section: Dna Binding/operator Releasementioning

confidence: 99%

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

2006

View full text Add to dashboard Cite

show abstract

“…The pioneering studies of Jacob, Monod, Ptashne, and Gilbert explained how these two processes, seeming inverses of each other, while being maintained in local chemical equilibrium, could still lead to robust genetic switches by coupling to protein synthesis and degradation, which are kinetically controlled far from equilibrium processes (1)(2)(3)(4). This classic picture, with the law of mass action at its core (5,6), suggests that understanding the molecular mechanism of the binding and release of transcription factors is of secondary interest compared with understanding the thermodynamics of protein-DNA recognition.…”

mentioning

confidence: 99%

Molecular stripping in the NF-κB/IκB/DNA genetic regulatory network

Potoyan

Zheng

Komives

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

119

View full text Add to dashboard Cite

Genetic switches based on the NF-κB/ IκB/ DNA system are master regulators of an array of cellular responses. Recent kinetic experiments have shown that IκB can actively remove NF-κB bound to its genetic sites via a process called "molecular stripping." This allows the NF-κB/ IκB/ DNA switch to function under kinetic control rather than the thermodynamic control contemplated in the traditional models of gene switches. Using molecular dynamics simulations of coarse-grained predictive energy landscape models for the constituent proteins by themselves and interacting with the DNA we explore the functional motions of the transcription factor NF-κB and its various binary and ternary complexes with DNA and the inhibitor IκB. These studies show that the function of the NF-κB/ IκB/ DNA genetic switch is realized via an allosteric mechanism. Molecular stripping occurs through the activation of a domain twist mode by the binding of IκB that occurs through conformational selection. Free energy calculations for DNA binding show that the binding of IκB not only results in a significant decrease of the affinity of the transcription factor for the DNA but also kinetically speeds DNA release. Projections of the free energy onto various reaction coordinates reveal the structural details of the stripping pathways. T he binding and release of protein transcription factors from DNA are fundamental molecular processes by which genes are regulated in the cell. The pioneering studies of Jacob, Monod, Ptashne, and Gilbert explained how these two processes, seeming inverses of each other, while being maintained in local chemical equilibrium, could still lead to robust genetic switches by coupling to protein synthesis and degradation, which are kinetically controlled far from equilibrium processes (1-4). This classic picture, with the law of mass action at its core (5, 6), suggests that understanding the molecular mechanism of the binding and release of transcription factors is of secondary interest compared with understanding the thermodynamics of protein-DNA recognition. The recent discovery of proteininduced release of a DNA-bound transcription factor in the NF-κB=IκB=DNA genetic switch changes this picture (7). The induced process, called "molecular stripping," opens up the possibility of molecular kinetic control of binding and release, thus overturning the classic paradigm based only on thermodynamic control. In this paper, we use molecular dynamics simulations of coarse-grained but predictive energy landscape models of the proteins along with their interacting DNA to explore first how the NF-κB transcription factor binds individually both to DNA and to its inhibitor IκB and then to study how an approaching IκB can strip the NF-κB from a DNA molecule to which it has already been bound, by forming an intermediate ternary complex. These simulations show that each of the binary binding events involves conformational selection of different NF-κB global conformations. Molecular stripping then occurs that is induced by forming the ternar...

show abstract

“…The actinomycin D molecule ( Fig. 1) contains two cyclic pentapeptide linkages, and a limited analogy may be drawn with the behavior of protein repressors (4). The interaction of actinomycin D and DNA has been extensively studied, as reviewed by Sobell (5) and Reich and Goldberg (6).…”

mentioning

confidence: 99%

Association of Actinomycin D and Deoxyribodinucleotides as a Model for Binding of the Drug to DNA

Krugh

1972

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The association of actinomycin D and eleven different deoxyribodinucleotides were studied as model complexes for the interaction of actinomycin D and DNA. All of the deoxyribodinucleotides containing guanine will complex with actinomycin D, and the guanine base has a preferred orientation with respect to the chromophore of actinomycin D (8). The association of actinomycin D and DNA may be monitored by observation of changes in the visible spectrum of actinomycin D when a complex is formed (9). These spectral changes may also be observed by the formation of complexes of actinomycin D and a wide variety of nucleosides and nucleotides (10). Actinomycin D showed a preference for guanosine or guanosine-5'-monophosphate, but the equilibrium constant is 1000 times less than for the interaction of actinomycin D and DNA (10' against 106).Hamilton, Fuller, and Reich (11) proposed that actinomycin D interacts with DNA primarily through hydrogen bond formation, with the chromophore located on the outside of the helix. Muller and Crothers (12) later proposed that the actinomycin D chromophore intercalates into the DNA helix. Sobell et al. (13) have solved the crystal structure of a complex of deoxyguanosine and actinomycin D, and have proposed a model (14, 15) for the actinomycin D-DNA complex that includes both intercalation and hydrogen bond formation. The guanine 2-amino group forms a strong hydrogen bond with the carbonyl oxygen of the L-threonine residue and a weaker hydrogen bond connects the guanine 1911 N(3) ring nitrogen with the N-H group on the iLthreonine residue in this model. The stoichiometry of the complex is one actinomycin D and two deoxyguanosines. In the crystalline complex, the nucleotides and the cyclic pentapeptides are related by an approximate 2-fold symmetry. The importance and possible implications of this 2-fold symmetry have been discussed in detail (5).I have investigated actinomycin D-DNA interaction by studying the association of actinomycin D and various deoxydinucleotides. As will be shown below, the deoxydinucleotides exhibit a great deal of specificity in their interaction with actinomycin D. These experiments support the model proposed by Sobell and Jain (14). MATERIALS AND METHODSActinomycin D was a gift of Merck, Sharpe and Dohme. The deoxydinucleotides were purchased from Collaborative Research, Inc., and, except for pdG-dG, were used without additional purification. The pdG-dG was dissolved in 5 mM phosphate buffer (pH 7.4) and passed through a Millipore filter. Guanosine-5'-monophosphate was purchased from 0 of C CH3

show abstract

Isolation of the Lac Repressor

Cited by 614 publications

References 11 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

Molecular stripping in the NF-κB/IκB/DNA genetic regulatory network

Association of Actinomycin D and Deoxyribodinucleotides as a Model for Binding of the Drug to DNA

Contact Info

Product

Resources

About