Ion Concentration and Temperature Dependence of DNA Binding:  Comparison of PurR and LacI Repressor Proteins†

Biochemistry ◽  
2001 ◽  
Vol 40 (27) ◽  
pp. 8109-8117 ◽  
Author(s):  
Markos I. Moraitis ◽  
Han Xu ◽  
Kathleen S. Matthews
Keyword(s):  
Dna Binding ◽  
Temperature Dependence ◽  
Ion Concentration ◽  
Repressor Proteins ◽  
Laci Repressor

scholarly journals Structural similarity in the DNA-binding domains of catabolite gene activator and cro repressor proteins.

1982 ◽  
Vol 79 (10) ◽  
pp. 3097-3100 ◽  
Author(s):  
T. A. Steitz ◽  
D. H. Ohlendorf ◽  
D. B. McKay ◽  
W. F. Anderson ◽  
B. W. Matthews
Keyword(s):  
Dna Binding ◽  
Structural Similarity ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Repressor Proteins ◽  
Cro Repressor

scholarly journals The Energetics of Molecular Adaptation in Transcriptional Regulation

2019 ◽  
Author(s):  
Griffin Chure ◽  
Manuel Razo-Mejia ◽  
Nathan M. Belliveau ◽  
Tal Einav ◽  
Zofii A. Kaczmarek ◽  
...  
Keyword(s):  
Free Energy ◽  
Transcriptional Regulation ◽  
Dna Binding ◽  
Coarse Graining ◽  
Binding Domain ◽  
Biophysical Model ◽  
Model Parameters ◽  
Biophysical Parameters ◽  
Binding Domains ◽  
Laci Repressor

Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only a subset of the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions.SummaryWe present a biophysical model of allosteric transcriptional regulation that directly links the location of a mutation within a repressor to the biophysical parameters that describe its behavior. We explore the phenotypic space of a repressor with mutations in either the inducer binding or DNA binding domains. Using the LacI repressor in E. coli, we make sharp, falsifiable predictions and use this framework to generate a null hypothesis for how double mutants behave given knowledge of the single mutants. Linking mutations to the parameters which govern the system allows for quantitative predictions of how the free energy of the system changes as a result, permitting coarse graining of high-dimensional data into a single-parameter description of the mutational consequences.

scholarly journals Predictive shifts in free energy couple mutations to their phenotypic consequences

2019 ◽  
Vol 116 (37) ◽  
pp. 18275-18284 ◽  
Author(s):  
Griffin Chure ◽  
Manuel Razo-Mejia ◽  
Nathan M. Belliveau ◽  
Tal Einav ◽  
Zofii A. Kaczmarek ◽  
...  
Keyword(s):  
Free Energy ◽  
Dna Binding ◽  
Binding Domain ◽  
Model Parameters ◽  
Free Energies ◽  
Epistatic Interactions ◽  
Binding Domains ◽  
Quantitative Accuracy ◽  
Laci Repressor ◽  
At Will

Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find that the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor ofEscherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions.

scholarly journals A Study of the CopF Repressor of Plasmid pAMβ1 by Phage Display

2000 ◽  
Vol 182 (10) ◽  
pp. 2973-2977 ◽  
Author(s):  
Emmanuelle d'Alençon ◽  
S. Dusko Ehrlich
Keyword(s):  
Dna Binding ◽  
Phage Display ◽  
Transcriptional Repressor ◽  
Dna Interaction ◽  
Filamentous Phage ◽  
Repressor Proteins

ABSTRACT We studied DNA binding of a transcriptional repressor, CopF, displayed on a filamentous phage. Mutagenesis of a putative helix-turn-helix motif of CopF and of certain bases of the operator abolished the protein-DNA interaction, establishing the elements involved in CopF function and showing that phage display can be used to study repressor proteins.

scholarly journals The Drosophila P-element KP repressor protein dimerizes and interacts with multiple sites on P-element DNA.

1996 ◽  
Vol 16 (10) ◽  
pp. 5616-5622 ◽  
Author(s):  
C C Lee ◽  
Y M Mul ◽  
D C Rio
Keyword(s):  
Dna Binding ◽  
Metal Binding ◽  
Molecular Mechanisms ◽  
P Element ◽  
Binding Motif ◽  
Mobile Dna ◽  
Repressor Protein ◽  
High Affinity ◽  
Repressor Proteins ◽  
Interaction Regions

Drosophila P elements are mobile DNA elements that encode an 87-kDa transposase enzyme and transpositional repressor proteins. One of these repressor proteins is the 207-amino-acid KP protein which is encoded by a naturally occurring P element with an internal deletion. To study the molecular mechanisms by which KP represses transposition, the protein was expressed, purified, and characterized. We show that the KP protein binds to multiple sites on the ends of P-element DNA, unlike the full-length transposase protein. These sites include the high-affinity transposase binding site, an 11-bp transpositional enhancer, and, at the highest concentrations tested, the terminal 31-hp inverted repeats. The DNA binding domain was localized to the N-terminal 98 amino acids and contains a CCHC sequence, a potential metal binding motif. We also demonstrate that the KP repressor protein can dimerize and contains two protein-protein interaction regions and that this dimerization is essential for high-affinity DNA binding.

Biological roles and mechanistic actions of co-repressor complexes

2002 ◽  
Vol 115 (4) ◽  
pp. 689-698 ◽  
Author(s):  
Kristen Jepsen ◽  
Michael G. Rosenfeld
Keyword(s):  
Dna Binding ◽  
Histone Deacetylase ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Protein Complexes ◽  
Genetically Engineered ◽  
Repressor Proteins ◽  
Multiple Protein ◽  
Genetically Engineered Mice ◽  
Cell Signaling Pathways

Transcriptional repression, which plays a crucial role in diverse biological processes, is mediated in part by non-DNA-binding co-repressors. The closely related co-repressor proteins N-CoR and SMRT, although originally identified on the basis of their ability to associate with and confer transcriptional repression through nuclear receptors, have been shown to be recruited to many classes of transcription factor and are in fact components of multiple protein complexes containing histone deacetylase proteins. This association with histone deacetylase activity provides an important component of the mechanism that allows DNA-binding proteins interacting with N-CoR or SMRT to repress transcription of specific target genes. Both N-CoR and SMRT are important targets for cell signaling pathways, which influence their expression levels, subcellular localization and association with other proteins. Recently, the biological importance of these proteins has been revealed by studies of genetically engineered mice and human diseases such as acute promyelocytic leukemia (APL) and resistance to thyroid hormone(RTH).

scholarly journals The role of subunits in yeast DNA-dependent ribonucleic acid polymerase A

1979 ◽  
Vol 181 (2) ◽  
pp. 301-308 ◽  
Author(s):  
C S Cooper ◽  
R V Quincey
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Temperature Dependence ◽  
Ribonucleic Acid ◽  
Specific Activity ◽  
Calf Thymus Dna ◽  
Calf Thymus

The properties of RNA polymerase A, which lacked the subunits of 48 000, 37 000 and 16 000 mol. wt., were compared with those of RNA polymerase A by using native calf thymus DNA as the template. The results showed that: (1) the specific activity of RNA polymerase A was about one-third that of RNA polymerase A; (2) more than 80% of RNA polymerase A, but only about 25% of RNA polymerase A, made RNA; (3) initiation by RNA polymerase A, but not by RNA polymerase A, began after a lag of 2 min; (4) the temperature-dependence for productive binding to DNA was greater for RNA polymerase A; (5) the apparent Km for UTP was greater for RNA polymerase A. These results support the supposition that the subunits missing from RNA polymerase A are involved in DNA binding [Huet, Dezélée, Iborra, Buhler, Sentenac & Fromageot (1976) Biochimie 58, 71-80] and show also that the loss of these subunits affects the elongation reaction.

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